Category Archives: DISCUSSIONS

Young Researchers — The Ethical Challenge

Rebecca Ram

Never before has there been a better opportunity for young researchers
to focus on replacement alternatives, not only to save animals
from unnecessary pain and suffering,
but also to pave the way for a career in cutting-edge innovation

Introduction

The Lush Prize awards and encourages individuals or organisations who have contributed to the global initiative to end animal testing, in the fields of science, public awareness, lobbying and training, and also aims to support young researchers who wish to develop a career in animal-free toxicology. The total yearly prize fund is £250,000. The ‘Young Researcher’ category of the Lush Prize welcomes nominations from early-career scientists who are keen to progress in research without animal testing. The award offers four £12,500 bursaries, to reward research and development specifically in methods for the entire replacement of animals in toxicity testing.
In this article, Lush Prize refers to testing without the use of animals by using the term ‘non-animal’ methods. They are validated, scientifically robust methods of safety testing in their own right. The use of terms such as ‘alternatives’ or ‘replacement’ methods (while useful for clarity sometimes) may suggest that animal testing is the ‘gold standard’ of safety testing, when much of the scientific industry, along with a wealth of research evidence, confirms that, aside from the suffering involved, animal tests do not reliably predict human responses. In addition, the term ‘alternatives’ is also used to describe the Three Rs methods,1 previously summarised as follows:
Refinement: to minimise suffering and distress to animals;
Reduction: to minimise the number of animals used; and Replacement: to avoid the use of living animals. Whilst reduction or refinement methods are positive steps, they are not achievements according to the ethos of the Lush Prize. We consider only the final ‘R’ (Replacement) to be a genuine alternative, as the other two Rs still involve animal use.

A brief review of previous findings

To discuss the relevance of the Young Researcher prize, a number of key animal protection organisations were contacted and interviewed during previous research for the Lush Prize. Some of these contacts are quoted and provide useful updates throughout this article. The key messages are:

— Although the acceptance and recognition of new technologies is growing at an encouraging rate, animal-free toxicology is still the ‘less travelled’ path, and any early-career researcher trying to progress in this area is likely to meet at least some resistance or challenges along the way. That said, the environment for the discussion of alternatives to animal use is expanding, so it is vital to be persistent and remain true to one’s values in pursuing career and networking opportunities.
— A very proactive attitude is needed; ethical scientists must actively seek out their opportunities, but the rewards can be hugely successful.
— Continuing to promote the anti-animal testing message to all relevant individuals in the young/early-career researcher field, as well as communicating this message at an earlier stage of education, is vital.

One of the key positive findings from interviews with previous Young Researcher prize winners is that “Young scientists don’t always have the prejudices about animal testing being the ‘best’ way of doing things”.2 An unbiased view and fresh perspective on cutting-edge science is essential, combined with raising awareness earlier in the educational system. There is also a strong link between the Young Researcher and Training prizes, as the latter awards are relevant to those involved in the education of a number of audiences, from children in early-stage schooling, through GCSE/A-level, to the undergraduate and postgraduate levels and beyond. There is considerable scope for early-career researchers working in a broad range of scientific or technical fields to get involved in non-animal methods. This is coupled with the fact that the in vitro toxicity testing market is projected to be worth $17,227 million by 2018.3 The EU cosmetic testing ban has played a key part in driving this growth.

Mainstream funding and development of Replacement methods

The importance of funding offered by the Lush Prize continues to grow. This is especially relevant to the Young Researcher Prize, as the bursaries awarded will directly fund the development of methods to replace the use of animals in ‘frontline’ research. As highlighted in previous research conducted for the Lush Prize, lack of finance is a major obstacle to the availability of non-animal methods. The ongoing reliance on animal research means that it continues to receive the vast majority of the available funding. Furthermore, those interested in non-animal research, not only have to maintain the momentum on their specific ideas and methods, but also face a need to continually look for funding or sponsorship, which ultimately impacts on the amount of time they directly spend on their research, as acknowledged by PETA in an interview with Lush Prize in 2012: “…people may have good ideas about non-animal methods, but they’re continually going to be seeking support…and funding for those”.4
To provide some figures to illustrate the above points, in the UK in 2012–2013, over £300 million of public funding was spent on projects which  “include an element of animal use”.5In contrast, a sum of just under £9 million was awarded to Three R projects broadly termed as ‘alternatives’, with the NC3Rs awarding £7 million of this total.5 The NC3Rs state that, of the funding they provide, “around 55 per cent of research awards are directed primarily at replacement, 25 per cent for reduction and 20 per cent for refinement”.6            Therefore, within this £9 million, a much lower sum was awarded to genuine non-animal (Replacement) methods, as a significant amount of ‘alternatives’ funding is donated to the other two Rs, which still involve animal use. For example, previous NC3Rs funding includes projects which develop scales for recognising facial expressions of pain in monkeys7 or facial grimace scales in rabbits.8 To add further perspective, since it was established in 2004, the NC3Rs has awarded just over £37 million in project funding. Based on the above figures, this equates to 12% of just one year’s Government funding of projects which include animal research.
Research carried out by the BUAV in 2013 revealed the stark lack of funding devoted to alternatives to animal testing across the EU Member States. Just
€18.7 million were devoted to methods relating to  the Three Rs in 2013, by only seven countries, with most Member States failing to assign any funding at all, and half of them not responding to the survey. Given that the available figures for 2011 show the total combined annual science R&D (research and development) budget for the EU to be almost €257 billion, the amount spent on alternatives is wholly inadequate, equating to just 0.007% of the total expenditure.9
These disappointing figures demonstrate the importance of independent funding for non-animal research, such as that which the Lush Prize offers. At its launch in 2012, the £50,000 total prize money for the Young Researcher Prize was allocated to five potential winners. This has now changed to award four prizes of £12,500, in order to increase the funding awarded to each individual, whilst still recognising the work of several researchers. Feedback from previous prize winners has indicated that these bursaries provide a meaningful amount, so the slight increase in funding across four awards will be of even more benefit, to go toward both research expenses and the cost of consumables.

Current and ongoing opportunities for keen young researchers

Banning animal testing will stifle innovation?

The claim that a ban on animal testing would stifle innovation was regularly made by industry as the 2013 EU cosmetics testing ban came into effect.10 Far from impeding research, the ban (both the 2009 and 2013 phases) had the opposite effect, and was the direct driver for the launch of new research into non-animal methods through large-scale, multinational projects (e.g. ReProTect11) as these two critical deadlines approached. R&D on new methods is innovation in itself, and it provides the perfect opportunity for those who genuinely want to be involved in cutting-edge, next-generation science, without causing animal suffering. The EU has led the way in progress on the development of alternative methods of testing to animals, and is considered ‘a leader in innovation’, something which should be reflected in the opportunities it offers young researchers and emerging graduate scientists.

Toxicity testing is toxicity testing, regardless of purpose

The validated and accepted non-animal (replacement) toxicity testing methods that are now available have been developed largely due to the phased EU ban on the animal testing and marketing of cosmetic ingredients and finished products. As a result, discussions on the replacement of animals in toxicity testing are far more common and perhaps are considered more acceptable in the cosmetics field. However, young researchers working or studying in other areas of toxicity may feel less able to speak out about their research interests, especially if they involve replacement/non-animal methods, as these are seen as more controversial than ‘two Rs’ (reduction or refinement) approaches. It is therefore important to recognise that these methods are now of essential use in other chemical testing sectors, such as the food or pharmaceutical industries. This demonstrates that when a non-animal method is developed and accepted, it can potentially be applied to the testing of any substance, for any purpose. This may encourage young researchers to voice their interests in the development and use of non-animal methods.
This is especially relevant as, despite the development of alternatives for use in areas such as cosmetics, toxicity testing in animals continues in many other industries. For example, in the UK in 2013, over 375,000 toxicity tests (from a total 4.12 million procedures) were performed on animals (mice, rats, rabbits, guinea-pigs, dogs, cats, monkeys, birds and fish).12 Another important point with regard to the Young Researcher Lush Prize, is that almost half of all animal experiments in the UK are carried out at  universities. One of the most concerning findings is that the increase in the use of genetically-modified animals (mainly mice) and the increasing use of zebrafish are, in some contexts, being considered as ‘alternatives’. This was highlighted by FRAME in a report on the Home Office annual statistics on animal use in Great Britain in 2012.13
The 2011 EU figures14 showed that over 1 million animals (1,004,873, from a total of just under 11.5 million animals) were used in toxicity testing across the EU states in that particular year. Of these, 111,166 animals were used in tests that were not even required by law (categorised as ‘no regulatory requirements’). The  archaic and much criticised LD50/LC50 (lethal dose or lethal concentration test, which tests the amount of substance required to kill 50% of the animals tested) accounts for the majority of the animals used each year, along with other lethal tests (34%). The other main use is simply categorised as ‘other’ toxicology tests (22%), followed by chronic/sub-chronic toxicity and reproductive toxicity. There is no official figure for the number of toxicity tests still conducted on animals worldwide (from the estimated yearly total of 115 million animals15 used in all experiments), as many countries omit this information or do not even count the numbers of animals used. However, a revised  estimate by Lush Prize researchers puts the number of toxicity tests carried out on animals worldwide at almost 9.5 million (from a total 118 million animal experiments).16

After the 2013 marketing ban, much work is still to be done

Although the EU cosmetics legislation has been the major driver of the development of non-animal toxicity testing methods in recent years, there is still much more to be done. This is illustrated very clearly, given that “Over 80% of the world allows animals to be used in cruel and unnecessary cosmetics tests and these animal tested cosmetics can be purchased in every country across the globe.”17
The proposed 2013 EU marketing ban on animaltested  cosmetics did finally go ahead, though the European Commission had previously considered the possibility of delaying the deadline on the basis of recommendations that necessary but still missing’ alternative methods would take much longer to be developed. For example, estimates of another 5–9 years were proposed for methods for skin sensitisation and toxicokinetics to be developed, and possibly even longer for full replacement in these areas. No estimates were provided at all for when repeat-dose toxicity, reproductive toxicity or carcinogenicity tests on animals might be developed. These timelines were estimated in a report published by the Commission in 2011.18  In the three years since that time, aside from the introduction of the 2013 ban itself (which went ahead regardless of the lack of alternatives available, which was great news), further work had been ongoing in the areas of toxicity testing which still need development. For example, in 2013, the Joint Research Centre (JRC) published its EURL-ECVAM Strategy to Avoid and Reduce Animal Use in Genotoxicity Testing.19 Similarly, the five-year long NOTOX project,20 launched in 2011 and involving a network of scientific expertise from several countries, works “towards the replacement of current repeated dose systemic toxicity testing in human safety assessment”. NOTOX is part of a wider project , funded under the EU Seventh Framework Programme (FP7), known as SEURAT (Safety Evaluation Ultimately Replacing Animal Testing). This project combines the research efforts of over 70 European universities, public research institutes and companies, and regularly posts open vacancies and research opportunities.21 Of particular relevance is that SEURAT hosted a Young Scientists Summer School to discuss replacement of repeat-dose toxicity testing in animals.22

Focusing on key areas

As highlighted by EURL-ECVAM,23 a key factor in the development of non-animal methods is the integration of a number of alternative test methods into a ‘battery’ that successfully addresses a number of endpoints, especially those which are considered more complex or need to be considered in-depth. For example, several alternatives available for skin testing, examine how a substance may react in various stages of topical application, absorption, irritation or corrosion, and provide very targeted and quantitative results, especially when compared to a crude skin test in rabbits or guinea-pigs. Therefore, non-animal methods which are still under development or undergoing validation, or ‘gaps’ in the development of methods where the greatest use of animals still occurs, such as reproductive or chronic toxicity, could be helped by awarding the Lush Prize to young researchers to allow them to potentially channel their ideas or research themes for specific replacement projects and encourage them to specialise in key areas.
Never before has there been a better opportunity for young researchers to focus on replacement alternatives, not only to save animals from unnecessary pain and suffering, but also to pave the way for a career in cutting-edge innovation. As highlighted by the New England Anti-Vivisection Society (NEAVS) in a previous interview with Lush Prize, linking an early-career scientist’s research to increased income and sponsorship is key:
“I think the solution for graduate students who want  to do more progressive in vitro research is to find the granting agencies that will help bring money in [to an institution]. …The key to changing institutions is bringing in grant dollars. When someone who wants to develop in vitro alternatives can show that they can bring in million-dollar grants, then institutions are going to have to accept it. They’re not going to turn money away, even if they want to try to suppress a certain ideology”.24
What this means, in effect, is that, if a researcher has ideas, but can also say “if you fund me, I propose to cut your costs, save you time, increase income and improve your business”, whilst this might be viewed as a challenge, their proposals are much more likely  to be considered.

Challenges for ethical early-career scientists

As previously highlighted, there remain ongoing prejudices toward switching from animal to non-animal research. Resistance to change, combined with ‘comfort’ in repeating accepted, conventional  methods, allows the animal research industry to maintain the status quo, despite ever-increasing recognition that animal testing is a flawed, overrated and outdated system. It must also be noted that the industry has, for decades, consisted of a network, not only of researchers, but also breeders, suppliers and transporters of animals across the world, who rely on animal testing to continue. There are other factors to consider — for example, some scientists (especially senior-level researchers) have based their entire careers on the use of animals, and are unable or unwilling to consider anything else; they may view switching to non-animal research as  the daunting and unattractive option of ‘starting again’. This may also apply to earlier career individuals, who have followed the mainstream route into animal-based toxicology to progress their careers to date, for example, since leaving university. This is echoed by several previous prizewinners, who felt that the undergraduate level of their education was the most challenging arena in trying to avoid the use of animals. Nevertheless, one positive finding from interviews with previous Young Researcher Prize winners is that “Young scientists don’t always have the prejudices about animal testing being the ‘best’ way of doing things”.2
Finally, to provide some useful insight into the types of scholarships available to young researchers, Appendix 1 gives a summary of 15 PhD studentships recently funded by the UK NC3Rs. A full list is shown to illustrate the types of research being undertaken
— however, it must be noted that the studentships cover the broader remit of the Three Rs, rather than the ‘replacement only’ criterion that the Young Researcher Prize demands.

Download the full article here (including Appendix 1) 

Rebecca Ram
Lush Prize
ECRA
41 Old Birley Street
Manchester M15 5RF
UK
E-mail: rebecca@lushprize.org

References and Notes

1 Russell, W.M.S. & Burch, R.L. (1959). The Principles of Humane Experimental Technique, 238pp. London, UK: Methuen.
2 Anon. (2012). Lush Young Researchers Prize 2012 — Research Paper, 22pp. Available at: http://www. lushprize.org/wp-content/uploads/2012/06/Lush-
Young-Researchers-Prize-2012-Research-Paper.pdf (Accessed 05.11.15).
3 Anon. (2014). In-Vitro Toxicology Testing Market worth $17,227 Million by 2018. Available at: http://www. prnewswire.co.uk/news-releases/in-vitro-toxicologytesting- market-worth-17227-million-by-2018-25358636 1.html (Accessed 05.11.15).
4 Interview with PETA, 20 August 2012.
5 Willetts, D. (2014). Hansard Written Answers. Animal Experiments: Business, Innovation and Skills written question — answered on 11th March 2014. Available at: http://www.theyworkforyou.com/wrans/?id= 2014-03-11a.188641.h (Accessed 05.11.15).
6 NC3Rs (2014). Funding schemes. Available at: http:// www.nc3rs.org.uk/landing.asp?id=27 (Accessed 06. 06.14).
7 NC3Rs (2014). Quantifying the behavioural and facial correlates of pain in laboratory macaques. Available at:  https://www.nc3rs.org.uk/quantifyingbehavioural-
and-facial-correlates-pain-laboratorymacaques
(Accessed 05.11.15).
8 NC3Rs (2012). The Rabbit Grimace Scale — A new method for pain assessment in rabbits. Available at: https://www.nc3rs.org.uk/news/rabbit-grimacescale-% E2%80%93-new-method-pain-assessment-rabbits
(Accessed 05.11.15).
9 Taylor, K. (2014). EU member state government contribution to alternative methods. ALTEX 31, 215– 218.
10 Anon. (2013). Europe Bans Marketing of Cosmetics Tested on Animals. Available at: http://ensnewswire. com/2013/03/11/europe-bans-marketing-o
f-cosmetics-tested-on-animals/ (Accessed 05.11.15). 11 Schwarz, M. (2011). Meta Analysis of a Battery Test of Reproductive Toxicity Assays: The ReProTect Experience. [Presentation given at Open Tox, Munich,
9–12 August, 2011.] Available at: http://www.opentox.org/meet/opentox2011/talks/OpenTox2011_
Talk-Schwarz.pdf (Accessed 05.11.15).
12 Home Office (2013). Annual Statistics of Scientific Procedures on Living Animals — Great Britain 2013, 59pp. London, UK: Her Majesty’s Stationery Office. Available at: https://www.gov.uk/government/ uploads/system/uploads/attachment_data/file/3278 54/spanimals13.pdf (Accessed 05.11.15).
13 Hudson-Shore, M. (2013). Statistics of Scientific Procedures on Living Animals 2012: Another increase in experimentation — Genetically-altered animals dominate again. ATLA 41, 313–319.
14 Anon. (2013). Report from the Commission to the Council and the European Parliament. Seventh Report on the Statistics on the Number of Animals Used for Experimental and Other Scientific Purposes in the Member States of the European Union. COM (2013) 859 final, 14pp. Brussels, Belgium: European Commission. Available at: http://eur-lex.europa.eu/
legal-content/EN/TXT/PDF/?uri=CELEX:52013DC085
9&from=EN (Accessed 05.11.15).
15 Taylor, K., Gordon, N., Langley, G. & Higgins, W. (2008). Estimates of worldwide laboratory animal use in 2005. ATLA 36, 327–342.
16 Anon. (2014). The 2014 Lush Prize: A Global View of
Animal Experiments 2014, 42pp. Available at: http://
www.lushprize.org/wp-content/uploads/Global_
View_of-Animal_Experiments_2014.pdf (Accessed 05.11.15).
17 Cruelty Free International (2012). Did you know animal tested cosmetics are for sale in every country in the world? Available at:  ttp://www.crueltyfree
international.org/en/the-issue (Accessed 06.06.14).
18 Adler, S., Basketter, D., Creton, S., Pelkonen, O., van Benthem, J., Zuang, V., Andersen, K.E., Angers- Loustau, A., Aptula, A., Bal-Price, A., Benfenati, E.,
Bernauer, U., Bessems, J., Bois, F.Y., Boobis, A., Bran – don, E., Bremer, S., Broschard, T., Casati, S., Coecke, S., Corvi, R., Cronin, M., Daston, G., Dekant, W., Felter, S., Grignard, E., Gundert-Remy, U., Heinonen, T., Kimber, I., Kleinjans, J., Komulainen, H., Kreiling, R., Kreysa, J., Leite, S.B., Loizou, G., Maxwell, G., Mazzatorta, P., Munn, S., Pfuhler, S., Phrakonkham, P., Piersma, A., Poth, A., Prieto, P., Repetto, G., Rogiers, V., Schoeters, G., Schwarz, M., Serafimova, R., Tähti, H., Testai, E., van Delft, J., van Loveren, H., Vinken,
M., Worth, A. & Zaldivar, J.M. (2011). Alternative (nonanimal)
methods for cosmetics testing: Current status and future prospects — 2010. Archives of Toxicology 85, 367–485.
19 Corvi, R., Madia, F., Worth, A. & Whelan, M. (2013). EURL ECVAM Strategy to Avoid and Reduce Animal Use in Genotoxicity Testing, 48pp. Ispra, Italy: European Commission, Joint Research Centre, Institute for Health and Consumer Protection. Available at: http://publications.jrc.ec.europa.eu/repository/bitstream/
111111111/30088/1/jrc_report_en_34844_on line.pdf (Accessed 05.11.15).
20 NOTOX (undated). Welcome to NOTOX. Available at: http://www.notox-sb.eu/ (Accessed 05.11.15).
21 SEURAT (undated). Welcome to the SEURAT-1 website. Available at: http://www.seurat-1.eu/ (Accessed 05.11.15).
22 SEURAT (undated). SEURAT-1 & ESTIV Joint Summer School — 8–10 June 2014. Egmond aan Zee, Netherlands. Available at: https://www.eurtd.com/
seurat-1/2014/summer-school/ (Accessed 05.11.15).
23 Anon. (2013). EURL ECVAM Progress Report on the Development, Validation and Regulatory Acceptance of Alternative Methods (2010–2013). Available at: http://ihcp.jrc.ec.europa.eu/our_labs/eurl-ecvam/
eurl-ecvam-releases-2013-progress-reportdevelopment-
validation-regulatory-acceptancealternative- methods (Accessed 06.06.14).
24 Interview with NEAVS, 20 August 2012.

A new approach to optimise the use of animal models in drug discovery through Big Data Sharing

Jiaqi Lu and Jianfei Wang

Sharing full details of animal models would enhance
consistency of models between establishments and
reduce the numbers of animals used for set-up and validation

With the continued rapid growth of Information technology (IT) and Cloud technology, the  concept of so called ‘Big Data’ has emerged over the last decade. Right now, this buzzword-phrase is being heard and talked about much more in the pharmaceutical industry and health care sectors.1–3 As a result of increased investment and better supply of information, pharmaceutical companies have recently been collating years of research and development data, including in vitro and animal preclinical test information, into medical databases. Meanwhile, the governments and other public stakeholders have been opening their vast stores of health-care knowledge, including data from clinical trials and information on patients acquired through public medical insurance programmes. 4,5 In parallel, some cross-boundary large IT companies, such as Google and Apple, have also jumped into this hot-tub and expect financial returns from the accelerating value and innovation in the health care and drug discovery industries.6,7

Historically, drug discovery and development has been a relatively isolated endeavour, with little information sharing evident among different pharmaceutical companies and academic researchers. In recent years, however, it has become apparent that pharmaceutical R &D is suffering from declining success rates and stagnant pipelines. This provides the impetus and an opportunity to change the landscape of drug discovery by utilising Big Data information. The current ability to generate and store vast amounts of information has led to an abundance of data and a growth in the discipline of Systems Pharmacology. Data are generated from several stages in the drug discovery process, including pre-clinical animal tests and Phase I, II and III trials, as well as post-marketing monitoring.8 Effectively utilising these data will help pharmaceutical companies to better identify new potential drug candidates and to develop safe, effective, approved medicines more quickly. In this article, a new approach to optimising the use of animal models in drug discovery through Big Data is explored.

Challenges and opportunities

Economic pressures, perhaps more than any other factor, are driving the demand for Big Data analysis and applications in drug discovery; this is appropriate, as it costs more than one billion US dollars to test and develop one new drug, and it often takes far more than ten years. In the early pre-clinical stage, the increasing costs of high-throughput screening of compound candidates, and of safety and efficacy studies in animal models, are major financial challenges to all pharmaceutical companies.

Animal model data are an important part of drug discovery Big Data — however, primary data generated from drug discovery animal research are infrequently accessed or re-used remotely from where they were generated. There is limited access to detailed drug discovery animal model data, since full information about the protocols has not always been published in research papers. This has led to a drive by many journals to enhance the depth of details given in the Methods sections  of manuscripts. Without this attention to detail, there have been missed opportunities for the continuous optimisation and  improvement of scientific methods and enhancement of innovation.

On the other hand, with the strengthening social pressures to avoid the use of laboratory animals in drug discovery, pharmaceutical companies and academia are finding it hard to demonstrate the application of the Three Rs principles to the satisfaction of the public. The sharing of full details of the animal models used in drug discovery would enhance the consistency of models between establishments and reduce the numbers of animals used to set up and validate the models.

Last, but not least, changing the mind set about confidentiality is a big challenge for all public and private organisations. Pharmaceutical R&D has always been a ‘secretive’ activity, conducted within the confines of the R&D department, with little external collaboration. Unless it is possible to identify an ideal future state with non-competitive aims, there is little value to investing in improving Big Data sharing capabilities. Today, public–private partnerships still represent a concept to be tested — therefore, if a new approach of sharing the full details of animal models used in drug discovery demonstrated its value and reduced attrition, then there would be an enlarged space for future developments in sharing information.

Landscape and strategy

The sharing and cross-analysis of pre-competitive drug discovery animal model information across public research organisations, pharmaceutical companies, Three Rs organisations, biotech companies and contract research organisations (CROs), through a one-stop sharepoint, would contribute to the simplification of the partners’ operating systems, would facilitate the delivery of more products of value through reduced attrition, and would enable all the partners to build trust by demonstrating their commitment to the Three Rs.

This one-stop sharepoint would allow data circulation within and beyond the original partnership. By enhancing interdisciplinary scientific reviews, animal studies could be optimised. Raw data, including cross-therapeutic animal models and protocols, drugvehicle effects, and positive and negative study results, would permit the assessment of the positive predictive value of each animal model. In addition, new information and hypotheses generated from data cross-analysis could be available to all partners, which would maximise the value of the animal research data. This non-competitive animal model information, such as guidelines on contemporary best practice and innovative alternative approaches to animal research, could also contribute to public knowledge and enhance animal welfare. In the end, by exchanging this information, all the partners would bolster external collaborations within and beyond the original partnership (Figure 1).
Figure 1

The importance of partnerships

The key component to achieving this goal is through partnership. No matter how public  research organisations, pharmaceutical companies, Three Rs organisations, CROs and biotech companies break the silos by enhancing collaboration with external partners,
all stakeholders can extend their knowledge and data networks through partnership.

Ideally, the following objectives could be achieved: identifying and discontinuing the use of animal models that are not sufficiently robust or fail to translate in the clinic; optimisation of the design and validation of animal models and protocols (e.g. to improve translation of animal models between laboratories or decrease model severity, to facilitate the informed choice of animal models, to share best practices and Three Rs advances, and to reduce duplication of efforts).

Academic partners could share insights from the latest scientific breakthroughs in cross-therapeutic animal models and make a wealth of innovation available. Normally, academia is willing to help improve the transfer of animal models between laboratories and the intra-laboratory reproducibility. Collaborations between the pharmaceutical companies could quickly identify and discontinue the use of animal models that identify treatments that fail to translate into efficacious medicines in the clinic. This could then lead to optimising the design/validation of animal models and protocols as a next step. This partnership could reduce clinical attrition, which would, in turn, reduce the financial cost of whole drug discovery process. Through collaboration with Three Rs organisations in order to learn best practices and Three Rs advances, stakeholders would enhance animal welfare by direct innovation and implementation of non-animal alternatives where they are the most needed, or by refining animal models to decrease severity and variability. Maximising external collaborations with CROs and new biotech companies could quickly add to or scale up internal capabilities and provide access to expertise in advanced technologies and animal models which would otherwise require establishment in-house.

Although this pattern of collaboration appears to be a win–win situation for all the partners, belief in the benefits of the data sharing culture and active participation still needs to be inspired. All stakeholders would have to recognise the value of Big Data analysis and sharing and be willing to act on its insights; a fundamental mind-set shift for many and one that may prove difficult to achieve. Confidentiality issues would also continue to be a major concern, although new IT technology can readily enhance private information protection in the databases. However, stakeholders would still have to be vigilant and watch for potential problems, as the increasing amount of information on animal models that is becoming openly available has the potential to the misunderstood by the general public.

Perspective

Big Data sharing is a new approach to optimising the use of animal models in drug discovery. Sharing animal model information, such as protocols, study results (including drug-vehicle effects and positive and negative data) and translational outcomes, in a single cross-therapeutic platform that uses a standard data capture and common ontology framework, would permit the secondary analysis of multiple datasets.

This would lead to higher efficiency for the assessment of preclinical drug efficacy and pharmacokinetics, and would also reduce the welfare impact on animals. Ultimately, it will also facilitate the re-use of animal data in the wider and more complex scenario of drug R&D, by facilitating linkage with other datasets, such as safety assessment datasets, chemistry and pharmacokinetics and pharmacodynamics (PK/PD). Crosspharma
collaborations in animal research are identified as a high-impact opportunity for accelerating scientific innovation and improving scientific output in animal model research for drug discovery, and for more tangible contributions to the Three Rs ethical principles across all the pharmaceutical industry. Subsequently, animal test dataset sharing across multiple pharmas and some prominent CROs, would further permit the appropriate assessment of the value of animal use in drug discovery and would lead to a reduction in the numbers of animals used in this work. Finally, if the pool of animal datasets generated by pharmas/CROs were ultimately augmented by the experimental data from many prominent academic institutions, it would be possible to generate an animal test search engine similar to Google Scholar. In an ideal situation, any proposal to carry out an animal test or use a particular animal model, should start with a search to identify suitable assays and an assessment of the potential utility of the model. This could be done by viewing existing positive and negative animal test data, as well as easily contacting partners experienced in these assays for advice. In addition, further animal model optimisation could be performed or unnecessary animal tests could be prevented. This would ultimately reduce animal use and reduce drug discovery costs and would speed up the drug discovery process by affording a greater chance of successful translation of efficacy to the clinic.

Acknowledgments

This work was supported by Key Projects in the National Science & Technology Pillar Program (No.  2011BAI15B03). The authors wish to thank Dr David Tattersall and Cheng Gao for their valuable suggestions.
Dr Jiaqi Lu
Department of Laboratory Animal Sciences
GlaxoSmithKline, R&D China

Author for correspondence:
Dr JianFei Wang
Head, Laboratory Animal Sciences
GlaxoSmithKline, R&D China
2F, Building 3
898 Halei Road
Zhangjiang Hi-Tech Park
Pudong
Shanghai 201203
China
E-mail: jianfei.j.wang@gsk.com

References

1 Fabricio, F.C. (2014). Big data in biomedicine. Drug Discovery Today 19, 433–440.
2 Marx, V. (2013). The big challenges of big data. Nature, London 498, 225–260.
3 Groves, P., Kayyali, B., Knott, D. & Kuiken, V.S. (2013).   The ‘big data’ revolution in healthcare: Accelerating value and innovation, 19pp. Center for US Health System Reform, Business Technology Office, McKinsey & Company.
4 SOTP (2012). Obama Administration Unveils “Big Data” Initiative: Announces $200 Million in New R&D Investments, 4pp. Washington, DC, USA: Office of Science & Technology Policy, Executive Office of the President, White House. Available at: https://www. whitehouse.gov/sites/default/files/microsites/ostp/big _ data_press_release_final_2.pdf (Accessed 19.09.15).
5 NIH (2015). NIH-led effort launches Big Data portal for Alzheimer’s drug discovery. Bethesda, Maryland, USA: National Institutes of Health. Available at: http://
www.nih.gov/news/health/mar2015/nia-04.htm (Accessed 19.09.15).
6 Harris, D. (2015). Google, Stanford say big data is key to deep learning for drug discovery. Houston, TX, USA: Knowingly, Corp. Available at: https://gigaom.com/ 2015/03/02/google-stanford-say-big-data-is-key-todeep-learning-for-drug-discovery/ (Accessed 19.09.
15).
7 Anon. (2015). Apple HealthKit. Cupertino, CA, USA: Apple Inc. Available at: https://developer.apple.com/ healthkit/ (Accessed 19.09.15).
8 Zhang, J., Hsieh, J.H. & Zhu,  H. (2014). Profiling animal toxicants by automatically mining public bioassay data: A Big Data approach for computational toxicology.
PLoS One 9, e99863.

Development and Validation of a Low-fidelity Simulator to Suture a Laparotomy in Rabbits

Juan J. Pérez-Rivero, Tonantzin Batalla-Vera and Emilio Rendón-Franco

An easily constructed, low-cost simulator
is assessed for its efficacy in the surgical training
of veterinary science undergraduates

Download a pdf of this article

Introduction

There is a growing need for the development of alternatives to reduce, replace and refine  the use of animals for surgical training in contemporary veterinary education at the undergraduate level. In the present study, a simulator to suture a midline laparotomy in the rabbit was designed, that could be constructed from widely-available and low-cost materials. The simulator was used to develop surgical skills in students at the undergraduate level of veterinary medicine. Thirty-five, third-year veterinary students, with no previous surgical experience, were divided into two groups: a control group that did not use the simulator (n = 19), and an experimental group that used the simulator three times to practise the suturing of a laparotomy (n = 16). Later, both groups performed
a midline laparotomy in an anaesthetised rabbit, and the rate of closure of each anatomical plane (peritoneum, additional reinforcement, and skin) was measured.

The usefulness of simulators

The surgical training of undergraduate students by using live animals provides few opportunities for real training and is applicable only to certain surgical techniques. In addition, it also raises serious ethical and animal welfare considerations. The students themselves are also subjected to a level of stress, this being, in most cases, a cause of errors. Consequently, they do not adequately benefit from the training provided.1,2

In veterinary medicine and animal sciences, the Three Rs principles are being implemented as widely as possible. This involves the reduction, replacement and refinement of animal use, both in experiments and in teaching.3 One way to accomplish this is through the use of various simulators in their different forms, such as synthetic simulators, multimedia simulations, virtual reality, carcasses, and ethically sourced animal tissues.4,5 These provide training alternatives, which permit the acquisition of skills to successfully meet the needs of future clinical and surgical experiences with live patients, and to ensure that maximum educational value is achieved during practical training.6

The fidelity of a simulator is determined by how much realism is provided through characteristics such as visual cues, touch, the ability to feedback, and interaction with the student. In general, simulators can be divided into two groups: high-fidelity simulators, which are usually highly technical, detailed and realistic; and low-fidelity simulators, which have a low level of realism, are usually made with widely available and low-cost materials, are often portable, and can be used on a table. Despite their simplicity, the latter group of simulators assist the development of psychomotor skills.7 Some limitations of the use of simulators are related to their cost or difficulty in sourcing spare parts. Moreover, despite the large number of simulators that have been developed, few studies have been conducted to evaluate their effectiveness, leaving the concept of teaching through simulators at an empirical stage.8 Therefore, it is necessary to develop inexpensive, easy-to-construct simulators that support the process of surgical teaching, and also to quantitatively assess the effectiveness of their use. Therefore, the aim of this work was to develop and validate a low-fidelity simulator to assist in the teaching of the correct technique for closing a rabbit midline laparotomy.
Simulator assembly

Development of the simulator

A 10cm long and 4cm wide opening was made in an empty plastic 500ml solution bottle (Pisa Agropecuaria, Guadalajara, Jalisco, Mexico), leaving protruding areas to represent both the xiphoid process and the pubic symphysis (Figure 1a). To give support to the bottle, an internal  cardboard lining was added, as well as three 3ml syringes widthwise (Figure 1b). Two 3mm thick silicone sheets were made by pouring 270ml of PE53® silicone rubber (Poliformas Plasticas, Mexico City, Mexico) into a mould, 23cm long by 13cm wide, which was allowed to set at room temperature for 24 hours.

The back-board from a standard paper clipboard was used as the simulator base, with the plastic bottle placed onto the board and the first sheet of silicone overlaid, in order to simulate the peritoneum (Figure 1c). Subsequently, the rectus abdominis muscles were simulated by placing two sheets of 3mm thick × 28cm long × 21cm wide polypropylene around the bottle (Foamy; Mylin, Mexico City, Mexico), leaving a gap of 3cm in width along the entire midline. Finally, this layer was covered with the second sheet of silicone to simulate skin, and both sheets of silicone were tightened onto the clipboard base with paper clips (Figure 2a). The appropriate size head, thorax and abdominal organs were fashioned from cotton fabric and added to the simulator prior to use (Figure 2a).
General view

Simulator validation

Thirty-five students in the third year of a veterinary medicine and zootechnics course at the Universidad Autónoma Metropolitana, Unidad Xochimilco, with no previous experience in surgery, received a 120-minute theory session, supported with slides, on the midline laparotomy technique and suture in rabbits.9 This was part of the Surgical-Veterinary Therapeutic Bases module. Later, the students were divided into two groups: the experimental group (n = 16), which  was organised in four surgical teams of four participants each, and the control group (n = 19), which was divided in four groups of four participants and one three-participant group. Each student was assigned his/her rotation within the group, in such a way that they all covered all the roles once (surgeon, first assistant, scrub nurse, and anaesthesiologist). Each surgeon/first assistant team (according to the assigned rotation) of the experimental group used the laparotomy simulator two days prior to the practice on the live animals. They were asked to repeat three times the following procedure: put the surgical drapes in place (Figure 2b); perform a 7cm incision, including all the layers of the simulator; suture, with continuous stitches, the first silicone layer (peritoneum), which was reinforced with inverted ‘U’ stitches; and suture, with Sarnoff stitches, the second silicone layer (skin). The first assistant was only allowed to help the surgeon in handling the surgical instruments that were used. The closure of planes was performed by using nylon 2-0 suture (Figure 3).
Simulator in use

Subsequently, the participants of both groups performed midline laparotomies on 35 clinically healthy New Zealand rabbits (Oryctolagus cuniculus), suturing midline (peritoneum) with continuous stitching, reinforcing (muscular fascia) with inverted ‘U’ stitches, and suturing the skin with Sarnoff stitches, all performed under general anesthesia, according to the method previously described by Perez-Rivero and Rendón-Franco.10 Both the control group and the experimental group performed one surgery weekly. In total, evaluations were completed in 4 weeks (i.e. one week for each participant from each team).

Since the lengths of the incisions were different in all the cases, the rate of closure of each anatomic plane and all planes in total, was calculated as follows: the length of each incision was measured (in centimetres), and this was divided by the time (in minutes) taken to complete the suturing. The result was expressed in minutes per linear centimetre of incision (MLCI). During the whole process, each group was supervised by two professors and five assistant instructors.
Table 1

Statistical Analysis

Students having the prior role of first assistant, scrub nurse, and/or anaesthesiologist, would have previously observed and/or helped in the performance of the laparotomy. This could have resulted in an improvement in their performance when participating as actual surgeons. To rule out these effects, total MLCI values were compared among the members of each group, according to whether they acted as the surgeon in week 1, 2, 3 or 4, to ascertain whether there was a significant difference in their surgical proficiency, by using the one-way ANOVA with a significant value p < 0.05.

Once the effects of previous observation and/or assistance were ruled out, the MLCI values of each individual anatomical plane and the totals were compared between the control and the experimental groups, by means of the ANOVA test (significant value p < 0.05). All tests were performed by using the PAST® program.11

Ethical and animal welfare considerations

The present protocol was approved by the Comité Interno para el Cuidado y Uso de los Animales de Laboratorio (Internal Committee for the Welfare and Use of Laboratory Animals) from the Universidad Autónoma Metropolitana Unidad Xochimilco, with
reference number DCBS.CICUAL.02.10.

Results of simulator use

Comparisons of the proficiency of group members according to the week in which they acted the role of surgeon did not show a difference (p > 0.05), supporting the idea that observation and/or assistance did not improve technique. When comparing the MLCI values of each plane as well as total MLCI values between the control group and the experimental group, all were different (p < 0.05) with a higher rate of closure for the experimental group. The MLCI values of each group, as well as their comparisons, are shown in Table 1.

The experimental group performed the three planes of laparotomy suture in 5.34 ± 1.63 minutes per linear centimetre of incision (MLCI), compared to the control group that performed it in 7.03 ± 1.77 MLCI. This difference was significant (one-way ANOVA; p < 0.05) and showed that repeating the procedure three times with the simulator improved
suturing skills in a laparotomy.

 

Discussion

When comparing MLCI values among the participants of each group independently, and not presenting differences, it is evident that observing and/or helping during the procedure did not render psychomotor skills or abilities in the participants. The use of complementary strategies, such as the use of the simulator, is necessary for a student to develop manual dexterity and the instrument skills required for the successful application of sutures.1,6

On the other hand, the experimental group demonstrated better suture skills for the laparotomy in rabbits after performing three repetitions of the procedure in the simulator. These findings agree with those reported by Aggarwal,12 who found in his study that laparoscopic surgeons required two repetitions of a particular procedure in a simulator, in order to learn it. The simulator required them to hold the tissue, lift it up, place a clip, and then cut; for trainees, seven repetitions were required to learn to perform the same procedure. However, we have to take into consideration that this particular procedure would have a longer learning curve than performing a suture.

Conclusions

The results make evident the advantages of the use of simulators, when recommended as training devices for undergraduate students. However, these models should be considered as complementary tools in the teaching of surgical procedures, for they help in the acquisition of skills and abilities that lead to better performance in real patients, and eventually reduce the number of training events that require the use of live animals.13

More studies are required to determine the time and number of necessary repetitions in training with these bench simulators, in order to reach an adequate level of proficiency. Further work will also be needed to make the simulators more realistic, and to investigate ways in which to take maximum advantage of this training tool.

Author for correspondence:
Dr Juan J. Pérez-Rivero
Departamento de Producción Agrícola y Animal
Universidad Autónoma Metropolitana Unidad
Xochimilco
Calzada del Hueso 1100
Colonia Villa Quietud
Delegación Coyoacán 04960
Mexico City
Mexico
E-mail: jjperez1_1999@yahoo.com

References

1 Langebæk, R., Eika, B., Jensen, A.L., Tanggaard, L., Toft, N. & Berendt, M. (2012). Anxiety  in veterinary surgical students: A quantitative study. Journal of Veterinary Medical Education 39, 331–340.
2 Smeak, D.D. (2007). Teaching surgery to the veterinary novice: The Ohio State University experience. Journal of Veterinary Medical Education 34, 620–627.
3 Russell, W.M.S. & Burch, R.L. (1959). The Principles of Humane Experimental Technique, 238pp. London, UK: Methuen.
4 Martinsen, S. & Jukes, N. (2005). Towards a humane veterinary education. Journal of Veterinary Medical Education 32, 454–460.
5 Kumar, A.M., Murtaugh, R., Brown, D., Ballas, T., Clancy, E. & Patronek, G. (2001). Client donation program for acquiring dogs and cats to teach veterinary gross anatomy. Journal of Veterinary Medical Education 28, 73–77.
6 Valliyate, M., Robinson, N.G. & Goodman, J.R. (2012). Current concepts in simulation and other alternatives for veterinary education: A review. Veterinarni Medicina 57, 325–337.
7 Perez-Rivero, J.J. & Rendón-Franco, E. (2012). Experience of the use of table-top simulators as alternatives in the primary surgical training of veterinary undergraduate students. ATLA 40, P10–P11.
8 Schout, B.M.A., Hendrickx, A.J.M., Scheele, F., Bemel mans, B.L.H. & Scherpbier, A.J. (2010). Validation and implementation of surgical simulators: A critical
review of present, past, and future. Surgical Endoscopy 24, 536–546.
9 Griffon, D.J., Cronin, P., Kirby, B. & Cottrell, D.F. (2000). Evaluation of a hemostasis model for teaching ovariohysterectomy in veterinary surgery. Veterinary Surgery 29, 309–316.
10 Perez-Rivero, J.J. & Rendón-Franco, E. (2014). Cardiorespiratory evaluation of rabbits (Oryctolagus cuniculus) anesthetized with a combination of tramadol, acepromazine, xylazine and ketamin3. Archivos de Medicina Veterinaria 46, 145–149.
11 Hammer, Ø., Harper, D.A.T. & Ryan, P.D. (2001). PAST: Paleontological statistics software package for education and data analysis. Paleontología Electrónica 4, 1–9.
12 Aggarwal, R., Grantcharov, T.P., Eriksen, J.R., Blirup, D., Kristiansen, V.B., Funch-Jensen, P. & Darzi, A. (2006). An evidence-based virtual reality training program for novice laparoscopic surgeons. Annals of Surgery 244, 310–314.
13 Denadai, R., Oshiiwua, M. & Saad-Hossne, R. (2014). Teaching elliptical excision skills to novice medical students: A randomized controlled study comparing low- and high-fidelity bench models. Indian Journal of Dermatology 59, 169–175.

 

 

In vitro models to mimic the endothelial barrier

Laurent Barbe, Mauro Alini, Sophie Verrier and Marietta Herrmann

Microfluidic technologies permit the replication in vitro
of geometrical features essential for the homeostasis of
all vascularised tissues in vivo, including the contribution
of pericytes to the endothelial barrier

Introduction

A functional microvasculature is critical for the homeostasis of all vascularised tissues.  accordingly, several diseases are associated with alterations in the microvasculature. For example, tumour angiogenesis is a major factor in determining the burden of the
disease. Furthermore, the formation of new vessels by angiogenesis and vasculogenesis is critical in the restoration of tissue function in ischaemic diseases. In tissue engineering, sufficient neovascularisation and early vessel anastomosis is thought to be a prerequisite
for the integration of the implant. These conditions have been extensively studied in animal models. However, in vivo studies have several limitations, including species differences and limited possibilities for imaging and tracking cells in the living animal. They also do not permit high-throughput and multiplexing applications. The development of microfluidic models of microvasculature and the endothelial barrier could help to overcome these problems and, most importantly, would replace a significant amount of animal experimentation. Nevertheless, microfluidic science is still an evolving research field, and many models do not address the endothelial barrier in its full complexity — for example, taking into account the contribution of pericytes.

The contribution of pericytes to the endothelial barrier

Pericytes are vascular mural cells associated with microvessels. The most common definition of pericytes goes back to their localisation, embedded in the endothelial basement membrane.1 Besides sharing the basement membrane, close interactions between endothelial cells and pericytes have been described, such as peg–socket contacts representing tight-, gap- and adherence-junctions.2 The relationship between the two cell types, particularly the anatomy of the pericyte coverage, reflects the function of the individual tissues. In organs with high exchange rates of gas and metabolites, the  distribution of pericytes is such that diffusion is minimally hindered.1, 3 Pericytes play an important role in vessel stabilisation, and underlying molecular signalling pathways have been described. Here, signalling through angiopoietin and Tie2 is critical; mutation of either angiopoietin 1 or Tie2 leads to mid-gestational death by cardiovascular failure in mice.2 In angiogenesis, the recruitment of pericytes is required for the stability of newly-formed vessels. Various studies have identified platelet-derived growth factor B (PDGF-B) as a major chemoattractant for pericytes. 4  Capillary and arteriolar pericytes also play an important role in inflammatory processes by guiding extravasating leukocytes toward their target. 5  Pericytes are also involved in several disease states, including airway remodelling in chronic allergic asthma6 and stroke.7 The identification of mesenchymal
stem cells and progenitor cells at perivascular sites, and the subsequent isolation and characterisation of such cells, suggests that pericytes have a role as multipotent progenitors.8-10

Current animal models

The function of pericytes in physiologically and pathologically relevant situations has been studied in transgenic mouse models containing LacZ-expressing or fluorophore-expressing pericytes.5, 6, 11 The migration and stimulation of pericytes in different disease situations was subsequently studied by the administration of pro-inflammatory factors, or by  backcrossing mice to a transgenic disease mouse model.5, 12  Interactions between labelled leukocytes and pericytes have been studied by lethal irradiation and the subsequent injection of bone-marrow cells from GFP mice.5 In addition, specific knockout lines have been used to study specific components of the cell junction and signalling complexes involved in pericyte– endothelial cell crosstalk, e.g. Sparc-deficient and Ccn2-deficient mice.13,14 Various animal models have also been developed, for studying the basic mechanisms of angiogenesis and vessel sprouting, including the cornea model, the chick chorioallantoic membrane (CAM) model, matrigel plug assays, and the dorsal skin fold chamber.15

Current microvascular models

Many factors determine the phenotypes of cells in vivo, including cell–cell interactions, interaction with the surrounding extracellular matrix, and the influence of various paracrine factors. Current cell culture vessels (dishes, flasks) cannot recapitulate these
aforementioned complex interactions. However, in recent years, microfluidic technologies have shown the potential to more-closely mimic the cellular microenvironment, at both the spatial and temporal levels.16 Typical microfluidic systems have geometrical features ranging in size from tens to hundreds of microns, and can host single cells or millions of cells arranged in a 2-D or a 3-D fashion. Microfluidic devices can accommodate flow control and therefore induce shear stress, which is known to have significant effects on the endothelial layer.17 This shear stress is absent in classical culture dishes. Furthermore, microfluidic technologies enable the generation of gradients over long periods of time, and also permit the control of paracrine factors in complex co-culture systems.18

In the past, various strategies were pursued to generate perfused microvessels in vitro.19 Different  levels of complexity were achieved, ranging from straight or branched channels within a material such as polydimethylsiloxane (PDMS), toward more complex microvascular networks within extracellular matrix hydrogels. In order to study cell–cell and cell– matrix interactions, microfluidic chips were designed that comprised two or more parallel channels, allowing the seeding of different cell types or hydrogels next to each other.20-22 Chen et al. reported on an alternative approach to studying the interactions between endothelial cells and other cell types.23  Here, each cell type was seeded in a different layer of the microfluidic device, separated by porous membranes; perfusion was applied to the endothelial layer of the chip.23 The embedding of microvessels in a 3-D matrix, potentially mimicking the perivascular tissue, represents another level of complexity. Several groups have addressed this challenge by generating endothelial cell-aligned microchannels within a collagen hydrogel.24–28 Such a set-up also allows the incorporation of pericytes in the hydrogel, which might eventually reassemble at the perivascular site of the endothelial cell layer.24, 26, 28, 29

Conclusions

Microfluidic technologies permit the replication of geometrical features found in vivo (i.e. small channels mimicking capillaries). In addition, due to the small dimensions involved, screenings are much less costly and time-consuming, as compared to similar in vivo studies. This is particularly interesting for applications such as testing the delivery of drugs. For the in vitro models to be successful, it is critical to consider all parameters of the endothelial barrier, including cell–cell and cell–matrix interactions, and the surrounding perivascular tissue.

Acknowledgements

The research of the authors is supported by the AO Foundation and the 3R Research Foundation Switz erland (Reduction, Refinement and Replacement of animal experimentation).

Dr Laurent Barbe
CSEM
Bahnhofstrasse 1
7302 Landquart
Switzerland

Dr Mauro Alini
AO Research Institute
Clavadelerstrasse 8
7270 Davos Platz
Switzerland

Dr Marietta Herrmann
AO Research Institute
Clavadelerstrasse 8
7270 Davos Platz
Switzerland

Author for correspondence:
Dr Sophie Verrier
AO Research Institute
Clavadelerstrasse 8
7270 Davos Platz
Switzerland
E-mail: sophie.verrier@aofoundation.org

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Building vascular networks. Science Translational Medicine 4,160ps123.
19 Zervantonakis, I.K., Kothapalli, C.R., Chung, S., Sudo, R. & Kamm, R.D. (2011). Microfluidic devices for studying heterotypic cell–cell interactions and tissue specimen cultures under controlled microenvironments. Biomicrofluidics 5, 13,406.
20 Bischel, L.L., Young, E.W., Mader, B.R. & Beebe, D.J. (2013). Tubeless microfluidic angiogenesis assay with three-dimensional endothelial-lined microvessels.
Biomaterials 34, 1471–1477.
21 Jeon, J.S., Zervantonakis, I.K., Chung, S., Kamm, R.D. & Charest, J.L. (2013). In vitro model of tumor cell extravasation. PLoS One 8, e56910.
22 Zervantonakis, I.K., Hughes-Alford, S.K., Charest, J.L., Condeelis, J.S., Gertler, F.B. & Kamm, R.D. (2012). Three-dimensional microfluidic model for tumor cell
intravasation and endothelial barrier function. Proceedings of the National Academy of Sciences of the USA 109, 13,515–13,520.
23 Chen, M.B., Srigunapalan, S., Wheeler, A.R. & Simmons, C.A. (2013). A 3D microfluidic platform incorporating methacrylated gelatin hydrogels to study physiological
cardiovascular cell–cell  interactions. Lab on a Chip 13, 2591–2598.
24Chrobak, K.M., Potter, D.R. & Tien, J. (2006). Formation of perfused, functional microvascular tubes in vitro. Microvascular Research 71, 185–196.
25 Price, G.M., Wong, K.H., Truslow, J.G., Leung, A.D., Acharya, C. & Tien, J. (2010). Effect of mechanical factors on the function of engineered human blood microvessels in microfluidic collagen gels. Biomaterials 31, 6182–6189.
26 van der Meer, A.D., Orlova, V.V., ten Dijke, P., van den Berg, A. & Mummery, C.L. (2013). Three-dimensional co-cultures of human endothelial cells and embryonic stem cell-derived pericytes inside a microfluidic device. Lab on a Chip 13, 3562–3568.
27 Morgan, J.P., Delnero, P.F., Zheng, Y., Verbridge, S.S., Chen, J., Craven, M., Choi, N.W., Diaz-Santana, A., Kermani, P., Hempstead, B., López, J.A., Corso, T.N., Fischbach, C. & Stroock, A.D. (2013). Formation of microvascular networks in vitro. Nature Protocols 8,
1820–1836.
28 Zheng, Y., Chen, J., Craven, M., Choi, N.W., Totorica, S., Diaz-Santana, A., Kermani, P., Hempstead, B., Fischbach- Teschl, C., López, J.A. & Stroock, A.D. (2012). In vitro
microvessels for the study  of angiogenesis and thrombosis. Proceedings of the National Academy of Sciences of the USA 109, 9342–9347.
29 Bichsel, C.A., Hall, S.R., Schmid, R.A., Guenat, O. & Geiser, T. (2015). Primary human lung pericytes support and stabilize in vitro perfusable microvessels. Tissue Engineering. Part A [E-pub ahead of print.]

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Renewed concern about the welfare of laboratory primates

PiLAS staff writer

One of the key points about the Three Rs is that, as Russell and Burch emphasised in The Principles of Humane Experimental Technique,1 “the humanest treatment of animals, far from being an obstacle, is actually a prerequisite for successful animal experiments”. Indeed, they said, the “wages of inhumanity” are “paid in ambiguous or otherwise unsatisfactory experimental results”. The PiLAS article by Chandna et al. on the commonplace single housing of primates in US laboratories,2 is therefore a matter of great concern, for both scientific and humanitarian reasons. It even appears that, far from trying to solve the problem, the US Government may be trying to cover it up.

In the UK, the NC3Rs is putting a great deal of effort into improving the welfare of the non-human primates used for research,3 but one has to wonder what fundamental and significant changes have taken place since the Home Secretary of the time, Douglas Hurd, accepted all but one of 17 proposals put to him by FRAME and CRAE (Committee for the Reform of Animal Experimentation) on the day that the Animals Scientific Procedures Act 1986 came into force.4,5 One of the points highlighted by FRAME and CRAE6 was that: “The very nature of a primate is such that you cannot institutionalise it in the laboratory and have a healthy animal. A primate is such that isolating in itself is deleterious.”

The current initiatives of the NC3Rs deserve to be applauded, but why do non-human primates continue to be used in research and testing at all, and how relevant and reliable are the data obtained to the understanding and treatment of diseases? Commenting on a recent report that the cynomolgus macaque is resistant to doses of paracetamol that would be fatal in humans,7 FRAME’s Scientific Director, Gerry Kenna, said in FRAME News that “This new research raises significant concern about the scientific validity to humans of drug safety studies undertaken in primates. The use of non-human primates in non-clinical safety testing is ethically undesirable and, in view of the substantial cost of such studies, can be expected to increase, markedly, the cost of drug development. Such studies should be considered only if they can be shown to be scientifically justifiable and there are no valid alternatives.”

The FRAME News item concluded by saying that “More time and money should be invested in cell-based and computer models that would be more reliable.”

1 Russell, W.M.S. & Burch, R.L. (1959). The Principles of Humane Experimental Technique, 238pp. London, UK: Methuen.
2 Chandna, A., Niebo, M., Lopresti-Goodman, S. & Goodman, J. (2015). Single housing of primates in US laboratories: A growing problem with shrinking transparency. ATLA 43, P30–P33.
3 Anon. (2015). The Welfare of Non-human Primates, 16pp. London, UK: NC3Rs. Available at: https://www.nc3rs.org.uk/welfare-non-human-primates (Accessed 16.07.15).
4 Anon. (1987). The Use of Non-Human Primates as Laboratory Animals in Great Britain, 16pp. Nottingham, UK, and Edinburgh, UK: FRAME and CRAE.
5 Anon. (1987/88). Response of the Home Secretary to the FRAME/CRAE primates report. FRAME News 17, 6−9.
6 Chivers, D. (1984). Comment in discussion session on Laboratory Primates. In Standards in Laboratory Animal Management, pp. 272. Potters Bar, UK: UFAW.
7 FRAME. (2015). Non-human primates and drug testing. FRAME News 74, 3.

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Turning Apples into Oranges? The Harm–Benefit Analysis and How to Take Ethical Considerations into Account

Herwig Grimm

How can expected study benefits and animal harms be weighed
against each other? What is the unit and common currency
that allows this weighing to be performed?

Suppose you are a scientist, working in the field of oncology and using live animals in your studies. Furthermore, suppose you have an excellent track record, you are well respected in the research community, and you regularly publish in high-ranking journals. One day, a person that you have not met before, wants to see you and talk about your work. Despite the fact that your time is extremely scarce, you invite this very person to your office and postpone your work on a follow-up research proposal. It turns out that the person who wants to talk to you is a member of a major animal protection group. She asks you the following question: “I came across a project summary, published according to Article 43 of Directive 2010/63/EU. Knowing your research, I think it is your project. Can you ethically justify the use of animals in your work? What I mean is: Do the benefits really outweigh the harms? And which ethical considerations do you take into account?” The animal protectionist is actually asking something for which you should, in fact, be well prepared. Directive 2010/63/EU1 was transposed into the national laws of the EU Member States. In Article 38(2) of the Directive, it is emphasised that a harm–benefit analysis of any project involving the use of animals must be carried out, in order to assess whether the harms related to the project are outweighed by the expected benefits. Furthermore, the relevant passage stipulates that ethical considerations have to be taken into account in this assessment (emphasis added by the current author):

“The project evaluation shall consist in particular of the following: d) a harm–benefit analysis of the project, to assess whether the harm to the animals in terms of suffering, pain and distress, is justified by the expected outcome taking into account ethical considerations, and may ultimately benefit human beings, animals or the environment.”

Consequently, the question arises as to how the harm–benefit analysis can be carried out, and how the term “taking into account ethical considerations” might be understood in this context. The meaning of this term is of major importance, since it provides a legally binding basis for the approval or rejection of projects. In other words, everyone who aims to secure the authorisation of a project in the EU has to make sure that the expected benefits outweigh the harms, and the justification must take into account ethical considerations, whatever that in fact means.All this has to be done on legal grounds — it is not just some fancy idea of animal protectionists.
The harm–benefit analysis:
A challenge or mission impossible?

At present, and to my knowledge, it is not at all clear how to prove, in a transparent and objective manner, that the expected benefits of an experimental study outweigh the expected harm to the animals to be used. Moreover, the actual meaning of the term “ethical considerations” remains vague, to say the least. How can expected study benefits and animal harms be weighed against each other? What is the unit and common currency that allows this weighing to be performed? And can ‘ethics’ help to turn apples into oranges, so that only comparable weights are on the scales? Since the Directive does not provide any specifications on standards for the harm–benefit analysis and how to take ethical considerations into account, the passage invites the reader to speculate.

The working document on Project Evaluation and Retrospective Assessment (WD 2013),2 from September 2013, is only of limited help. It provides important criteria and ideas, but it leaves the reader without help when it comes to a methodology for transparent decision-making. It refers to the Bateson Cube, which indicates what should be taken into account, but whether its dimensions (i.e. benefit, likelihood of benefit, harm to animals) are ethical in nature, and whether these dimensions are sufficient for the harm–benefit analysis, remains open to question. Furthermore, no measure is provided to allow the various dimensions to be made comparable. Take, for example, ‘benefit’. Here, a set of analytic questions is given (WD 2013, p. 21):
What will be the benefits of the work?
Who will benefit from the work?
How will they benefit/impact?
When (where possible) will the benefits be achieved?

But, even if we had the answers to all these questions, how can they be integrated in the harm–benefit analysis? Does this mean that there should be no research on orphan diseases, because only few people can benefit? Does it mean that it matters who benefits in terms of age? Does this mean that a new cold remedy is more important than a new cancer treatment, since many more people will use it and it therefore has a greater impact? Is research more important, if it will bring about practical benefits sooner, and should this influence the harm–benefit analysis?

Similar questions arise on the harm side. Is the severity classification enough? Shall we add all harms done to individual animals, and if so, how do we deal with harms that are related to the project indirectly, such as harm to animals that were necessary to establish the particular mouse strain used? Is the absolute number of animals used in an experiment something that should be taken into account — or is it acceptable to adhere to the Three Rs criterion of reduction, and to use the minimum number of animals? And if the absolute number should count, what is a ‘high’ number (100 dogs, or 12,000 mice?) and does ‘high’ vary, depending on the research field in question? And if we knew all that and more, how could we bring all these criteria into one methodology, in order to carry out a transparent harm–benefit analysis? At the moment, this seems to be a mission impossible, rather than a challenge to be dealt with.

Steps to tackle the problem: Criteria, methodologies and committees

One could of course go on and on with this list of open questions. In order to answer at least some of them, researchers from various fields — and in particular, ethicists — try to take on the challenge. For example, the Messerli Research Institute (Vienna, Austria) hosted an international symposium in March 2013, in order to discuss possible steps toward overcoming the aforementioned problems. A conference on the harm–benefit analysis was also held in Bergen in 2014, and we debated the issues at the World Congress on Alternatives and Animal Use in the Life Sciences, held in Prague in late 2014. Many well-known experts in the field of ethical evaluation of animal experiments took part. At all these meetings, the aim was to bring together state-of-the-art knowledge with regard to the issues. For example, in Vienna, 22 speakers from eight European countries and the USA discussed their experiences and the current situation surrounding these issues in their respective countries. The lively discussions went to show that many challenges remain, but some issues can be solved.

In the course of the Vienna symposium, not only the criteria and aspects that should go into the harm–benefit analyses were debated. Importantly, different methodologies such as checklists, scoring systems or comparative methodologies, were also introduced. Most of the experts emphasised the importance of independent and well-balanced committees, and the integration of lay people (i.e. non-specialists) and representatives of animal welfare organisations into these committees. Taking into account the lay people’s perspectives and current public opinion would contribute to an up-to-date ethical evaluation of animal experiments. But, by taking all of these factors into account, are we any way nearer solving the problem of how to transparently weigh apples against oranges successfully?

Although no ‘super-theory’ to resolve all of the issues was identified, the challenges became much clearer. Furthermore, things to be avoided came to the table: A particular and major threat that has to be avoided when developing methodologies for the harm–benefit analysis became very clear, and that is over-bureaucratisation. Any methodology for the harm–benefit analysis has to be a user-friendly tool that leads to deeper reflection on individual animal experiments. The different forms of methodologies were summarised in the following three groups:

— comparative methodologies that use positive lists (white-lists) and negative lists (black-lists) of animal experiments, in order to evaluate the project in question;
— scoring strategies that quantify the extent to which relevant criteria are met, and that provide an algorithm for calculating the harms and benefits of projects; and
— check lists that provide binary (yes/no) evaluation methods, e.g. in the form of decision trees.

Whether these methods are used within or without the committee structure makes a big difference, and both scenarios are indeed possible. Ideally, applicants should follow a structured procedure and provide the relevant information according to a set of clear standards and criteria that have to be met. Interdisciplinary committees would then be able to evaluate the projects according to the same standards and criteria. These evaluations could inform the competent authority’s decisions.

Many things could be said, and indeed have been said in the past, about methodologies, and a great deal has also been written on the subject. Needless to say, we did not come to any final conclusions at the symposium in Vienna, nor in Prague, nor in Bergen, on this complex but vital matter.

How to proceed from here?

In order to reach a clearer vision of how the harm–benefit analysis can be brought into a feasible methodology, any ideas are welcome. Exchanging ideas and arguments might inspire and boost the debate. This short article serves as an open invitation to all interested experts in the field to start such a debate. Since this should happen in a focused way, the following topics might be useful to guide the discussion:

Committees and their limitations and advantages: A great number of EU Member States have established local and national committees to support the authorities in decision-making on submitted proposals. Certainly, such committees have the advantage of bringing skilled experts from the sciences, statisticians, representatives of animal protection groups and lay people, to work together in order to formulate a statement on harms and expected benefits. However, these committees often work without explicit methodology or criteria. So the question arises as to how they can safeguard transparent and non-arbitrary decision-making when they carry out harm–benefit analyses. I am sure that many of the readers are experienced members of such committees, and it would be very useful, if they would contribute with their experience, knowledge and ideas.
Methodologies: It would be of great interest to share knowledge on the advantages and disadvantages of methods used. If committees and the national authorities apply consistent methods and explicit criteria, it would be of utmost importance to get into an exchange of views and experience on whether and how such methods can support and improve the decision-making process.
Ethics and Law: A third question relates to the terminology used in the Directive. If ethical considerations should be taken into account, should these considerations exceed existing law or is “ethics” to be understood within legal limits (and is not allowed to exceed existing law)? Here, ethics runs the risk of contradicting the principle of legality in constitutional states. In other words: How is the term “taking ethical considerations into account” interpreted in different countries. It would be great to get some insight into this.
Experience from the past: Generally, since many countries have carried out harm–benefit analyses in the past, knowledge of their experiences could contribute to future developments.
Ideas for the future: Finally, a possible thought experiment is to think about where we are going to be in 20 years’ time. How will the debate look in 2035? Will we still be trying to weigh apples against oranges?

These questions and statements aim to initiate a debate that is relevant to all EU Member States and everybody involved in animal research. It would be very useful, if experts in this forum were willing to find some time to contribute to a lively and future oriented discussion, in order to solve at least some of the open questions mentioned above. The idea is to continue to build up knowledge on the process of harm–benefit analysis in animal research, and maybe improve the situation for both animals and researchers. Perhaps this forum could bring us closer to the point where researchers were able to respond to the question as to whether, and indeed which, projects involving live animals are justifiable, and which are not. Being able to respond to the question as to whether a project is worth carrying out or not, could demonstrate that scientists are able to take on this responsibility in a knowledge-based society and thus can contribute to ethical welfare.

Prof. Dr Herwig Grimm
Messerli Research Institute
Veterinary University of Vienna,
Medical University Vienna,
and University of Vienna
Veterinärplatz 1
1210 Vienna
Austria
E-mail: herwig.grimm@vetmeduni.ac.at

References
1 Anon. (2010). Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes. Official Journal of the European Union L276, 22.10.2010, 33–79.
2 Anon. (2013). National Competent Authorities for the Implementation of Directive 2010/63/EU on the Protection of Animals Used for Scientific Purposes. Working Document on Project Evaluation and Retrospective Assessment, 42pp. Brussels, Belgium: European Commission.

Download a pdf of the article here: Discussion Grimm.

Coffee in Class: An Alternative to Animal Experiments in Pharmacology?

Anoop Kumar Agarwal, Syed Ilyas Shehnaz, Razia Khanam and Mohamed Arifulla

The stimulant effect of coffee on psychomotor performance was
introduced as a potential alternative clinical pharmacology
experiment for medical and pharmacy students

Animal experiments have been designed and standardised to demonstrate the effects of certain drugs on body organs, as part of undergraduate health professional education. However, the logistics of animal availability, the expenses incurred, increasing awareness of concerns about animal welfare1–3 and the ‘Three Rs’ concept (i.e. Replacement, Refinement and Reduction),4 have often either reduced these experiments to tutor demonstrations or have resulted in their complete withdrawal from the undergraduate curriculum.5

As an alternative to satisfy the ethical concerns of animal rights activists, Computer Assisted Learning (CAL) was introduced.6–8 Although CAL is an effective means of fulfilling the educational objectives of laboratory sessions, the lack of hands-on experience with living tissues, the lack of practical experience to facilitate the future application of the procedures in research, as well as the absence of biological variation, are the major limitations of CAL.9

In view of the current scenario, we considered it necessary to investigate alternative exercises which would expose the students to experimental methodology with scientific explanation. Clinical pharmacology experiments, such as dosage calculations, rational drug selection, evaluation of drug information, and the analgesic effect of NSAIDs, have been used to supplement CAL.10 In an effort to identify alternatives to animal experiments and CAL at the undergraduate level, the Department of Pharmacology, Gulf Medical University, Ajman, United Arab Emirates, introduced a new experiment in the teaching curriculum of Bachelor of Medicine/Bachelor of Surgery (MBBS) and Pharm D (Doctor of Pharmacy) programmes. The aim of the experiment was to demonstrate the stimulant effect of coffee on psychomotor performance in students, by using simple paper and pencil tests, namely, the Six-Letter Cancellation Test (SLCT) and the Digit/Letter Substitution Test (DLST). These tests objectively assess the psychomotor functions of an individual, and integrate different mental functions, such as perception, recognition, integration and reaction, in the assigned task. These tests are not meant for assessing memory or intelligence. Since they are speed tests, performance is influenced by mental alertness, concentration and coordination abilities. Both tests consist of three sections: instructions, the key (target) letters, and the working-out part. In the SLCT, the subject identifies the key letters in the working-out part, whereas in the DLST, the numbers in the working-out part have to be substituted by the corresponding letters given in the key. The duration of each test is 90 seconds. In both the tests, the extent of the working-out part exceeds the potential for completion in the stipulated time. The maximum and minimum scores for these tests vary in different subgroups. Parallel worksheets (with a different key) are used on each occasion (Figure 1 and Figure 2), to nullify the effect of memory.11

Minimal materials are required

The equipment required for the experiment is readily available at low cost. It comprises three sets of parallel worksheets for the SLCT, three sets of parallel worksheets for the DLST, a stop watch, an office bell, and standard hot coffee (2g instant coffee/200ml).

Figure 1

The protocol

The experimental protocol is divided into five individual stages:

1. The practice session: The tutors familiarised themselves with the tests and planned the experiment to ensure smooth implementation. Ethical approval was granted from the Institutional Ethics Committee prior to the conduct of the experiment. The tests were administered during the pharmacology laboratory sessions to seven batches of 25–30 medical and pharmacy students. The students were told about the importance and relevance of the tests, and were issued with the instructions necessary for performing the tests. Written consent was obtained from those who volunteered to participate in the experiment. One practice session was organised, in order to familiarise the participants with both the tests.

2. The pre-coffee session: The worksheet for the SLCT were distributed, and the students were asked to write their names on the back of the sheet. This was fllowed by the first bell, indicating the beginning of the ‘working-out time’, which ended with a second bell after 90 seconds. The students were asked to start and stop immediately when the bell rang, and strict monitoring of time was ensured. The sheets were randomly exchanged among the students for score calculations. The second test, the DLST, was administered in a similar manner, after an interval of 5 minutes.

3. The post-coffee session: A 20ml cup of standard coffee was served to each student. After 20 minutes, the two tests were re-administered as before, with parallel worksheets, i.e. a new key was used in each session, to nullify the effect of memory.

4. Interpretation of scores: The students were asked to record the scores of the two tests in the three sessions (practice, pre-coffee and post-coffee) in their record books, and to draw conclusions based on a pharmacological explanation. The mean scores of both the tests for the practice, precoffee and post-coffee sessions were expressed as the mean ± standard deviation (SD). Comparison of scores was done by using the Wilcoxon signed-rank test. The significance level was set at 0.05.

5. Student feedback: Feedback on the experiment was obtained by using a structured, content-validated and pre-tested questionnaire on a five-point Likert-like scale (strongly agree to strongly disagree). The statements enquired about the ease of understanding and performing of the tests, the appropriateness of the time allowed and the methodology, the understanding of the concepts and the generation of links between theory and actual effects of CNS stimulants, the interest levels generated, and the willingness to perform similar experiments in the future. In addition, open-ended responses about the students’ experiences were also encouraged. Voluntary participation was emphasised, and full confidentiality of the data was ensured to all the
participants.

Agarwal table 1Figure 2

Results

Of a total of 180 medical and pharmacy 162 participated in the experiment (response rate 90%). The mean scores (± SD) of the SLCT for the practice, pre-coffee and post-coffee sessions are given in Table 1. Although there were no significant differences between the practice and pre-coffee session scores for both the tests, a statistically significant difference (p < 0.05) was observed between the pre-coffee and the post-coffee scores. Furthermore, a small number of students obtained a low score (5%), or had no change in their scores after coffee intake (6%).

The students gave a very good feedback on the experiment, as reflected in the questionnaire (Table 2). However, in the free responses, a few students (12%) reported that they lost interest by the third session, due to the simplicity and repetitive nature of the test, and that, as a result, they felt that they did not perform at their best.

Table 2

Discussion

We have endeavoured to introduce a clinical pharmacology experiment as an alternative to CAL and animal experiments, for use in undergraduate health professional education. This experiment has very few requirements, and can be easily performed within laboratory session time-frames. The experiment also reinforces the concept of the mild stimulant effect of coffee. However, test performance is affected by various factors, including motivation, understanding, interest, mood, environment, quality of the worksheet, and personality type, so scores obtained may vary for different groups and sub-groups. Moreover, a clear understanding of the principle of the paper and pencil test among the faculty is important, in order to plan the experiment in an organised manner. Although these tests had been used earlier as a teaching tool among medical students, the perceptions of the students had not been obtained.11 The experiment was reintroduced in our setting with certain modifications, and student feedback was obtained with regard to its acceptability and relevance. This experiment had been introduced for the first time in the Pharm D curriculum to demonstrate the effects of the drug.

The test scores of both the tests (SLCT and DLST) showed a significant increase in psychomotor performance after coffee intake, suggesting a stimulant effect. However, a number of sources of variation were identified: differences in quantity of coffee consumed, loss of interest due to repetitive nature of the test, and anxiety after coffee intake. The latter is a known side-effect of coffee in certain individuals. Students who did not show an increase in score stated that they frequently consumed coffee during the day.

The student feedback revealed that the majority found the experiment interesting and informative. This could probably motivate them to learn more about the drugs and their effects. However, in the open-ended responses, a few students felt that the tests were too simple and that a higher degree of complexity was necessary to keep up their interest.

Author for correspondence:
Dr Syed Ilyas Shehnaz
Department of Pharmacology
Gulf Medical University
PO Box 4184
Ajman
United Arab Emirates
E-mail : shehnazilyas@yahoo.com

References

1 Gitanjali, B. (2001). Animal experimentation in teaching: Time to sing a swan song. Indian Journal of Pharmacology 33, 71.
2 Solanki, D. (2010). Unnecessary and cruel use of animals for medical undergraduate training in India. Journal of Pharmacology & Pharmacotherapeutics 1,59.
3 Desai, M. (2009). Changing face of pharmacology practicals for medical undergraduates. Indian Journal of Pharmacology 41, 151–152.
4 Guhad, F. (2005). Introduction to the 3Rs (refinement, reduction and replacement). Contemporary Topics in Laboratory Animal Science 4, 58-59.
5 Setalvad, A.R.N. (2009). Medical Council of India, New Delhi, Amendment Notification of 8 July 2009 to the Minimal Standard Requirements for Medical Colleges with 150 Admissions Annually, Regulations 1999. Available at: http://www.mciindia.org/helpdesk/how_to_start/STANDARD%20FOR%20150.pdf (Accessed
01.08.14).
6 Dewhurst, D. (2004). Computer-based alternatives to using animals in teaching physiology and pharmacology to undergraduate students. ATLA 32, 517–520.
7 Badyal, D.K., Modgill, V. & Kaur, J. (2009). Computer simulation models are implementable as replacements for animal experiments. ATLA 37, 191–195.
8 Wang, L. (2001). Computer-simulated pharmacology experiments for undergraduate pharmacy students: Experience from an Australian University. Indian Journal of Pharmacology 33, 280–282.
9 Kuruvilla, A., Ramalingam, S., Bose, A.C., Shastri, G.V., Bhuvaneswari, K. & Amudha, G. (2001). Use of computer assisted learning as an adjuvant to practical pharmacology teaching: Advantages and limitations. Indian Journal of Pharmacology 33, 272–275.
10 Gitanjali, B. & Shashindran, C.H. (2006). Curriculum in clinical pharmacology for medical undergraduates of India. Indian Journal of Pharmacology 38, Suppl., 108–114.
11 Natu, M.V. & Agarwal, A.K. (1997). Testing of stimulant effects of coffee on the psychomotor performance: An exercise in clinical pharmacology. Indian Journal of Pharmacology 29, 11–14.

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Discussion Agarwal

‘One R’ is the new ‘Three Rs’

Craig Redmond

The Lush Prize, which rewards initiatives to end animal testing,

believes that more attention needs to be given to the ‘One R’

of absolute replacement, and that research methods that

exploit animals in any way (including tissues and cells)

should not be considered as ‘alternatives’

Introduction
Replacement of animal experiments is one of the Three R concepts (alongside Reduction and Refinement) first put forward by Russell and Burch in 1959.1 However, this can be either absolute replacement (i.e. methods that do not involve animals or animal tissues) or relative replacement (e.g. methods that use only cells or tissues of animals in vitro or ex vivo).

It has become accepted by many in the research community that some uses of animals can be classed as ‘alternatives’. In particular, the use of whole animals of species thought to either not experience pain or to have a lower level of sentience (e.g. fruit flies, nematodes and zebrafish), or of animal parts (including tissues, embryos, sera and cells). The use of these methods is reinforced by regulatory bodies, making it more difficult to reach a time when no animal use will occur in scientific research. André Ménache, of Antidote Europe, believes that in the region of 80% of ‘alternatives’ validated by ECVAM (the European Union Reference Laboratory for alternatives to animal testing) still use animals or animal tissues (personal communication, 04.09.13).

Founded in 2012, the Lush Prize rewards global initiatives to end animal testing, particularly in the area of toxicology. A total of £250,000 is shared annually between five prize categories covering science, training, young researchers, lobbying and public awareness. Lush Prize believes that more attention needs to be given to the ‘One R’ of absolute replacement, and that research methods that exploit animals in any way (including tissues and cells) should not be considered as ‘alternatives’.

Alternatives’ that still exploit animals
The United States Department of Agriculture refers to ‘alternatives’ as “a term that has different meanings to different people, and this difference largely depends on which side of the issue one is found”.2 So, for example, animal researchers might use relative replacement methods in addition to their use of animals (or look to refine existing animal tests), whereas abolitionists see ‘alternatives’ in terms of absolute replacement. Other examples of where the use of the term ‘replacement’ serves to reinforce the idea that relative replacement is routinely acceptable are:

— where Russell and Burch defined a replacement technique as “any scientific method employing non-sentient material which may in the history of animal experimentation replace methods which use conscious living vertebrates”.3 The words ‘non-sentient’, ‘conscious’ and ‘vertebrates’ ensure that the use of invertebrates and species considered as ‘lower organisms’ continues to be accepted.
— when, in its current “step-by-step approach to an alternatives search”, the Johns Hopkins University Center for Alternatives to Animal Testing (CAAT) suggests that, in addition to cell culture, tissue culture, models, simulations, etc., researchers “might look for a non-mammalian animal model
— fish or invertebrates, for example — that would still give you the data you need”. 4

Perhaps as a direct result of this widespread inherent acceptability of relative replacement alternatives, researchers at the University of British Columbia, looking into people’s acceptance of the use of particular species in laboratories, found that species such as fish and invertebrates “are typically rated below mammals, and, as such, are often considered an appropriate replacement for mammals in research”. 5

The philosopher Joel Marks notes that “developing alternatives to the use of animals can mean simply using a different animal” and considers that “the characterisation of the other animal […] as ‘lower’ on a phylogenetic ‘scale’ is arbitrary and disputed. The alternatives movement is therefore at risk of becoming a bait-and-switch con”.6 By this, Marks means that ‘alternatives’ are advertised as one thing (i.e. absolute replacement), but often turn out to be something completely different (i.e. simply the use of another species).

Some examples of relative replacement alternatives are:
Invertebrates: The horseshoe crab, Limulus polyphemus, is used in the Limulus amoebocyte lysate (LAL) assay. This method replaces the rabbit pyrogen test for the detection of endotoxin in, for example, hepatitis B vaccines. 7 The rabbit test involves injecting the test substance into a marginal vein of the ear of each of three rabbits.8 However, the LAL assay uses blood cells from the horseshoe crab, with up to 30% mortality resulting from the bleeding procedure.9

Fish: Zebrafish (Danio rerio) are widely used in research, including research on genetics, cancer and, increasingly, toxicology. The maintenance costs of zebrafish are less than one thousandth of the costs of maintaining mice,10 and they can produce 100–300 eggs per week, making their embryos useful for high-throughput screening.11 It is widely acknowledged that fish can feel pain, 12 with as much evidence for this as there is for birds and mammals.13 Other studies have shown that they have conscious awareness.14

Tissues: Hundreds of thousands of animals are bred and killed each year in Britain alone, solely to provide tissues for research.15 Human tissues to be preferred, due to species differences, yet animal tissue is often used on the grounds of cost and availability.16 Human tissue can be obtained from patients during diagnosis, removed as ‘waste’ during surgical operations, placentas or ‘afterbirth’, or tissues obtained after death.15

People can voluntarily donate blood or other tissue for transplantation or research, or their organs or bodies after death. Human tissue removed from the body in the course of disease diagnosis or treatment is the main source.17 However, although upwards of 600,000 residual surgical tissues are generated each year in England and Wales, only a tiny fraction of them are made available to researchers.18

The use of fetal calf serum
The move toward the use of in vitro cell culture to provide both human and animal cells for alternative methods is a step in the right direction. However, the use of fetal calf serum (FCS) as a cell culture media supplement is unacceptable, in the light of the availability of serum replacements and serum-free culture methods.19 collected for FCS production is obtained by cardiac puncture, performed by inserting a needle directly into the heart of the non-anaesthetised fetus.20 according to Jochems et al.20 it is very likely that the fetus is alive at the time of blood collection, and “will experience pain and/or suffering at the moment of heart puncture for blood collection and possibly for a period after that, until it actually dies”. The scientific validity of using FCS has been questioned. Risk of contamination is an issue,21 with the potential presence of viruses, bacteria, mycoplasma, yeast, fungi, immunoglobulins and endotoxins.20

Conclusions
These are just a few examples of animal use that some see as ‘alternatives’. The use of sentient animals, such as fish and horseshoe crabs, should not be accepted by those working in the field of alternatives to animal testing, despite the entrenched position within the research community and regulatory bodies. Neither should cruel processes such as the collection of FCS be condoned. In addition to greater humanity and greater acceptability, there are a multitude of clear scientific benefits to avoiding the use of animals or animal products.

The Lush Prize promotes the ‘One R’ of Replacement over all of the ‘Three Rs’, believing that the  true absolute replacement of animals is essential for ethical and scientific progress.

Craig Redmond
Lush Prize
Unit 21
41 Old Birley Street
Manchester M15 5RF
UK
E-mail: craig@lushprize.org
References
1 Russell, W.M.S. & Burch, R.L. (1959). The Principles of Humane Experimental Technique, 238pp. London, UK: Methuen.
2 Taylor Bennett, B. (1996). Alternative methodologies. In Essentials for Animal Research. A Primer for Research Personnel, pp. 9–17. Darby, PA, USA: Diane Publishing.
3 Balls, M. (1994). Replacement of animal procedures: Alternatives in research, education and testing. Laboratory Animals 28, 193–211.
4 Altweb Project Team (undated). Search for Alternatives. Baltimore, MD, USA: CAAT, Johns Hopkins
University. Available at: http://altweb.jhsph.edu/resources/searchalt/index.html (Accessed 22.10.14).
5 Ormandy, E.H., Schuppli, C.A. & Weary, D.M. (2012). Factors affecting people’s acceptance of the use of zebrafish and mice in research. ATLA 40, 321–333.
6 Marks, J. (2012). Accept no substitutes: The ethics of alternatives. The Hastings Center Report 42, Nov–Dec (Special Report), S16–S18.
7 Park, C.Y., Jung, S.H., Bak, J.P., Lee, S.S. & Rhee, D.K. (2005). Comparison of the rabbit pyrogen test and Limulus amoebocyte lysate (LAL) assay for endotoxin in hepatitis B vaccines and the effect of aluminum hydroxide. Biologicals 33, 145–151.
8 WHO (2013). The International Pharmacopoeia. Preface: 3rd Supplement. Geneva, Switzerland: World Health Organisation. Available at: http://apps.who.int/phint/en/p/docf/ (Accessed 07.09.13).
9 Leschen, A.S. & Correia, S.J. (2010). Mortality in female horseshoe crabs (Limulus polyphemus) from biomedical bleeding and handling: Implications for fisheries management. Boston, MA, USA: Massachusetts Division of Marine Fisheries. Available at: http://www.mass.gov/eea/docs/dfg/dmf/publications/mortality-in-female-horseshoe-crabsabstract.
pdf (Accessed 25.10.14).
10 Reed, B. & Jennings, M. (2011). Guidance on the housing and care of zebrafish Danio rerio, 64pp. Horsham, West Sussex, UK: Research Animals Department, Science Group, RSPCA.
11 van Vliet, E. (2011). Current standing and future prospects for the technologies proposed to transform toxicity testing in the 21st century. ALTEX 28, 17–44.
12 FAWC (1996). FAWC report on the welfare of farmed fish, 43pp. London, UK: Farm Animal Welfare Council.
13 Braithwaite, V. (2010). Do Fish Feel Pain?, 208pp. Oxford, UK: Oxford University Press.
14 Cottee, S.Y. (2012). Are fish the victims of ‘speciesism’? A discussion about fear, pain and animal consciousness. Fish Physiology & Biochemistry 38, 5–15.
15 Focus on Alternatives (undated). Focus on Human Tissue in Research, 4pp. Available at: http://www.frame.org.uk/dynamic_files/foa_humantissue.pdf (Accessed 03.09.13).
16 EMA (2012). Committee for Advanced Therapies (CAT) Scientific Workshop: Reducing the number of laboratory animals used in tissue engineering research — 11th October 2012 — European Medicines Agency, London [EMA/CAT/708346/2012], 4pp. London, UK: European Medicines Agency. Available at: http://www.ema.europa.eu/docs/en_GB/document_library/Report/2012/12/WC500136419.pdf (Accessed 25.10.14).
17 Nuffield Council on Bioethics (1995). Human Tissue: Ethical and Legal Issues, 182pp. London, UK: Nuffield Council on Bioethics. Available at: http://nuffieldbioethics.org/project/human-tissue/ (Accessed 25.10.14).
18 Bunton, D. (2011). The use of functional human tissues in drug development. Cell & Tissue Banking 12, Issue 1, 31–32.
19 Newman, C. (2003). Serum-free cell culture — the ethical, scientific and economic choice. The Biomedical Scientist, September 2003, 941–942.
20 Jochems, C.E., van der Valk, J.B., Stafleu, F.R. & Baumans, V. (2002). The use of fetal bovine serum: Ethical or scientific problem? ATLA 30, 219–227.
21 Focus on Alternatives (2009). Serum-free Media for Cell Culture, 52pp. http://www.frame.org.uk/dynamic_files/foa_fcs_free_table_may09.pdf
(Accessed 03.09.13).
 

The 9th World Congress on Alternatives and Animal Use in the Life Sciences

The 9th in this series of congresses which began in 1993, was held at the Hilton Prague Hotel, Czech Republic, on 24–28 August 2014. Its organisation was co-chaired by Dagmar Jirová (Prague) and Horst Spielmann (Berlin), on behalf of the Alternatives Congress Trust, with the administrative support of Guarant International.

The Congress was attended by about 1,100 participants, and the programme consisted of seven plenary lectures, more than 450 oral presentations, and about 500 posters. In addition, there was an exhibition with 60 booths, plus a number of satellite meetings and workshops, and many private discussion sessions.

The overall focus of the Congress was Humane Science in the 21st Century, as represented by nine main themes: new technologies; predictive toxicology; the Three Rs in academia and education; communication, dissemination and data sharing; efficacy and safety testing of drugs and biologicals; human relevance; ethics; refinement and welfare; and global co-operation, regulatory acceptance and standardisation.

The congress facilities provided by the hotel were superb, which helped to make this a particularly friendly congress. Many of the participants in the 1993 Congress were present, but it was also good to see a great number of younger scientists, 41 of whom had been specifically invited due to generous sponsorship.

It would be impossible to say much in detail about the Congress, given the enormous variety of topics covered. However, it is worth noting that two of the plenary lecturers gave contrasting insights into the state of humane science and the Three Rs as it is today.

Uwe Marx (Berlin) described the breathtaking progress being made toward developing a “human-on-a-chip”, as means of providing information of direct relevance to humans, replacing the need to resort to laboratory animal models. Early organ-on-a-chip versions — comprising artificial lungs, liver, kidneys, heart and gut — are already in use.

By contrast, Roman Kolar (Neubiberg) warned that many apparent commitments to the Three Rs have proved to be no more than lip-service, and political initiatives to avoid or replace animal experimentation have either failed dramatically, or have been watered down in the political decision-making process.

Of the Three Rs, it appeared that Reduction was rarely mentioned in Prague, and Refinement, however welcome, pales into insignificance in the face of the huge ethical and logistical dilemmas involved in maintaining animals under laboratory conditions. Replacement took the centre stage in most of the sessions, but, given the year-on-year increase in the production and use of, in particular, genetically-modified animals, there is a lot more to be done before humane science becomes more than just a dream. The 10th Congress will be held in Seattle in 2017 — it is to be hoped that much more progress will have been made by then.

Human Bioengineered Artery Models for In Vitro Atherosclerosis Research: Fact or Fiction?

Human bioengineered artery equivalents may represent a first
step toward the future replacement of the animal models
used for studying the initial phases of atherosclerosis.

According to the US National Center for Health Statistics, cardiovascular diseases — including coronary heart disease and cerebrovascular disease — are leading causes of death in the western world. Sixty eight percent of all cardiovascular diseases are related to atherosclerosis, resulting in a substantial morbidity and mortality, as well as a major socioeconomic burden.1 As a result of current demographic changes, namely, the significant increase in the elderly population, the burden of atherosclerosis is on the rise.2 It is therefore not surprising that research on atherosclerosis is becoming more and more important, and substantial financial resources are being transferred into this particular field of biomedical research. Alongside this development, the use of animal models in atherosclerosis research has also exponentially increased over the last few decades.
According to a MEDLINE search conducted in May 2014, 29,795 publications can be found when entering the search terms “atherosclerosis AND animal”. Assuming that only half would represent animal in vivo studies, and assuming that only 15 animals were used per study, then this would render a load of 223,463 animals used in atherosclerosis trials published in the MEDLINE database alone. Interestingly, the number of publications was stable until the beginning of the 1990s, at approximately 200 entries per year. However, starting at around 1990, a dramatic increase in publications is observed (see Figure 1), demonstrating the increase of research in this area of cardiovascular disease.

Figure 1
The total number of MEDLINE entries found was 29,795.  The number of papers published on this topic per year substantially increased after the 1980s.
In the current decade, this number has reached more than 1700 papers per year. [Data were retrieved from
www.ncbi.nih.gov on 25.05.2014.]

An overview of atherosclerosis
development

Atherosclerosis represents a chronic inflammatory disorder that is the underlying cause of most cardiovascular diseases.1–3 As a first event in the pathogenesis of atherosclerosis, LDL (a cholesterol-rich lipoprotein) accumulates in the intimal layer of the healthy vessel wall.4,5 As a next step, the activated endothelial layer promotes the adhesion and transmigration of monocytic/macrophagic blood cells, which themselves take up the accumulated LDL particles via a receptor-mediated process. This process is the basis for the formation of the initial ‘fattystreak lesion’ in the vascular wall.4,6 This lesion then serves as the basis for a progressive immunological reaction, leading to the formation of more and more complex, highly organised vascular lesions, which finally — after a number of years — result in a ‘vulnerable plaque lesion’.4,7 If these vulnerable plaque lesions rupture at the location of the ‘fibrous cap’, a thrombotic (potentially lethal) response is evoked. This may lead to the occlusion of vessels, such as in the case of myocardial infarction.8 So far, these two processes, i.e. lipoprotein accumulation and the transmigration of macrophages, have received the greatest attention with regard to achieving optimal mimicry of human atherogenesis in vitro.

Current animal models
According to the recent review by Getz et al.3, several different animal models are used for testing therapeutic agents and studying the etio-pathogenesis of atherosclerosis. Getz et al. state that no model is ideal, as each has its own advantages and limitations with respect to manipulation of the atherogenic process and modelling human lipoprotein profiles. Due to the relative ease of genetic manipulation and the short time-frame for the development of atherosclerosis, murine models are currently the mostextensively used animals in atherosclerosis research. However, murine atherosclerosis development significantly differs from human atherosclerosis.9 Therefore, a range of further larger animal models have been widely used, including rabbits, rats, guinea-pigs, hamsters, birds, dogs, swine and nonhuman primates (NHPs).3,9,10 In particular, the development of porcine11 and NHP models12–14 has been intensified in recent years. In pigs, especially the diabetic hypercholesteraemic model,6 the Rapacz-familial hypercholesteraemia model,11 the PCSK9-gain-of-function model and different mini-pig models for investigating metabolic syndrome11,14 were seen as important steps toward more-representative animal research.11 Predominantly, the more-human analogous genetic expression patterns, as well as the development of high-risk atherosclerotic lesions, were seen as major advantages of these transgenic porcine models.11 Also, NHPs have been used for atherosclerotic research12,15 — mainly rhesus and cynomolgous monkeys,12 African green monkeys,12,13 and baboons15 — as they provide unique opportunities to evaluate effects of long-term diets and/or pharmaceutical agents. Particularly, the aspect of dyslipidaemia profiles similar to those of humans seems outstanding in these models, as summarised by Chen et al.12: NHPs “… closely resemble humans in lipid metabolism and disease physiology compared to lower species.” Also, a large cross-species study revealed that only NHPs and certain dog species (e.g. obese beagle models) provide a close match to dyslipidaemic humans.12

This development toward higher (more-clinically relevant) animal species is associated with several problems. Besides the profound ethical considerations surrounding large scale animal research in higher species (dogs, pigs, NHPs), the financial hurdles associated with these experiments also limit further and broad-scale scientific progress. This is even more important when considering the results of large cross-species studies, which reveal that even these higher species show significant differences, and in particular, when it comes to research on lipid metabolism. 14

New in vitro models
Given the ethical and scientific obstacles associated with the animal models, the development of more representative in vitro models based on human cells seems to be of outstanding importance, and ultimately represents the only solution to this situation. In addition, such in vitro models could not only overcome the ethical discussions resulting from large scale animal experiments, they could also potentially allow for more broad-scale screening platforms, thus making atherosclerosis research more effective.

Therefore, given the lack of models that fully represent the human atherothrombotic pathology, several in vitro studies have been initiated that focus on the development of a human cell-based model for studying atherosclerosis ex vivo. These in vitro models hold the potential to significantly reduce and replace animal in vivo experiments and — at the same time — to increase the predictive value of the experiments with regard to human pathologies. Most of the initial experimental in vitro studies used two dimensional (2-D) cell culture systems (of endothelial cells or macrophages) to study basic mechanisms of atherosclerosis.16 In spite of the importance of these pioneering investigations, these 2-D models lacked both a) the three-dimensional (3-D) structure and b) the pulsatile blood-flow environment present in native human arteries. Therefore, these studies on 2-D culture dishes were (and are) associated with significant limitations, as they do not account for the complexity of the native artery environment, including all of the cell–cell and cell–matrix interactions that are present in vivo. Particularly, the signalling between the luminal endothelial cell component and the sub-endothelial vascular interstitial cell (media) component is missing. Given that vascular smooth muscle cell activation, proliferation, and migration have been identified as key processes of atherosclerosis development, the single cell nature or the 2-D aspect of these models represent major shortcomings.17 In order to address these limitations, several groups developed 2-D co-culture models of human and animal endothelial cells and vascular interstitial cells (smooth muscle cells).18–20 In these models, the two different cell types were separated by a preformed collagen matrix, which is intended to mimic the presence of the basal lamina, resembling for the first time a 3-D-like cell arrangement. However, these attempts were also limited by the non-physiological attachment of cultured cells to plastic dishes (or synthetic trans-well membranes) on the 2-D scale.16 In addition, in these types of co-culture systems, the culture times were too short for the development of cell–extracellular matrix interactions that are typical of the in vivo situation.16 Seeking further improvement, Dorweiler et al.21 were able to create one of the first long-term co-culture models through the use of fibrin gels. Besides an improvement in the overall culture duration, they also reported the accumulation of LDL and immune cells in the (bioartificial) sub-endothelial matrix, representing a significant improvement compared to previous systems. 21,22 However, in spite of the proof of lipoprotein accumulation, this non-dynamic co-culture system lacks the full 3-D cylindrical architecture of a native vessel. Even more importantly, it also lacks the haemodynamic pressure/flow/shear-stress environment present in the native situation, which plays a fundamental role in atherosclerosis development in vivo.

As part of therapeutic bioengineering approaches, several groups have successfully demonstrated the manufacture of tissue-engineered artery equivalents (with a 3-D cylindrical native-like architecture) under pulsatile, native-like flow (‘bioreactor’) conditions. These bioengineered vascular grafts showed a native analogous microstructure and approximation of the biomechanical behaviour of native tissues.23 These autologous tissue-engineered vascular grafts were originally designed for the surgical repair of congenital cardiac malformations. They were successfully implanted into large animal models, and exhibited adequate functionality in vivo for periods of up to 240 weeks.24,25 In an initial attempt to overcome the above-mentioned limitations, Robert et al.16 combined the technologies of atherosclerosis research with vascular bioengineering techniques. After creating a native-like human vascular cell-based layered bioengineered artery equivalent by using a pulsatile flow bioreactor system (also containing a basement membrane), LD- and HD-lipoproteins were integrated into the flow loop. The accumulation of cholesterol-rich lipoproteins represents a first, essential step in atherosclerosis development.5 In the engineered artery model, endothelial as well as sub-endothelial lipoprotein recovery of LDL, as well as HDL, could be observed over time. As a next step of human atherosclerosis development, lipoprotein-mediated endothelial activation leads to monocyte infiltration and transmigration.6 In the bioengineered artery model, monocytes injected into the flow loop were also found to adhere to the (activated) endothelium and to transmigrate into the vascular interstitium, which was comparable to the events that occur in the native counterpart.16 In spite of the several improvements on the previous 2-D and 3-D co-culture in vitro models, the proposed model only focused on short time-points. The formation of ‘foam’ cells, an integral part of atherosclerosis development, has not yet been demonstrated.

Summary and conclusions
In biomedical research, there has been an increasing interest over the last few years in the creation of systems for modelling human disease in vitro. The major difficulties encountered in these attempts stems from the complexity of native structures and processes, which are difficult, or sometimes impossible, to model in vitro. The resulting simplification of in vitro model systems holds the risk of limited, or even misleading, scientific output, with questionable representation of the native human situation. Atherosclerosis is an excellent example of this development, as it displays a highly complex multi-organmediated pathology. To make matters worse, atherosclerosis is not only a complex disease, but it is also very species-specific — indicating that animal research also has limited representative value. This scientific ‘dilemma’, combined with the astronomic health as well as socioeconomic impact of this disease, has recently stimulated the development of alternative in vitro models. What would be needed is a fully dynamically-perfused human native-analogous artery model, in order to study pathogenic processes in atherothrombosis development — processes which might harbour potential for therapeutic intervention. The latest advances in the field of biomedical engineering have led to the development of bioengineered artery models that overcome at least some of the limitations of previous models. However, these latest model systems suffer from extensive simplification, including: a) the use of cell culture media containing xenogenic and immunogenic elements (instead of blood); b) the use of low pressure conditions; c) the lack of defined shear-stress; d) extensive ex vivo cell expansion; and e) the lack of neurogenic as well as lymphogenic vascular elements. However, most importantly, the ‘vulnerable plaque lesion’ — which forms after years (or even decades) in humans — would be the most interesting structure to be studied, as this is the pre-stage to any potentially lethal, ruptured vascular plaque. Considering the short cultivation periods in the currently available bioengineered artery models (i.e. of only a few weeks), so far, the in vitro modelling of human atherogenesis has been limited to the study of the very initial phases of the disease.

Therefore, the in vitro mimicry of all phases of human atherosclerosis and vulnerable plaque development still represents ‘fiction’ rather than a scientific fact. However, the recent developments, as well as the rapid progress in bioengineering and biotechnology, create significant hope that the realisation of in vitro modelling of human atherosclerosis is within our grasp.

References
1 National Heart, Lung & Blood Institute (2012). NHLBI Fact Book, Fiscal Year 2012 — By Section, 201pp. Bethesda, MD, USA: National Institutes of Health, US Department of Health & Human Services. Available at:  http://www.nhlbi.nih.gov/about/factbook/toc.htm (Accessed 25.05.14).
2 Edo, M.D. & Andrés, V. (2005). Aging, telomeres, and atherosclerosis. Cardiovascular Research 66, 213–221.
3 Getz, G.S. & Reardon, C.A. (2012). Animal models of atherosclerosis. Arteriosclerosis,  Thrombosis, & Vascular Biology 32, 1104–1115.
4 Robert, J., Weber, B., Frese, L., Emmert, M.Y., Schmidt, D., von Eckardstein, A., Rohrer, L. & Hoerstrup, S.P. (2013). A three-dimensional engineered artery model for in vitro atherosclerosis
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6 Glass, C.K. & Witztum, J.L. (2001). Atherosclerosis: The road ahead. Cell 104, 503–516.
7 Rosenson, R.S., Brewer, H.B., Jr, Davidson, W.S., Fayad, Z.A., Fuster, V., Goldstein, J., Hellerstein, M., Jiang, X.C., Phillips, M.C., Rader, D.J., Remaley, A.T., Rothblat, G.H., Tall, A.R. & Yvan-Charvet, L. (2012). Cholesterol efflux and atheroprotection: Advancing the concept of reverse cholesterol transport. Circulation 125, 1905–1919.
8 Brokopp, C.E., Schoenauer, R., Richards, P., Bauer, S., Lohmann, C., Emmert, M.Y., Weber, B., Winnik, S., Aikawa, E., Graves, K., Genoni, M., Vogt, P., Lüscher, T.F., Renner, C., Hoerstrup, S.P. & Matter, C.M. (2011). Fibroblast activation protein is induced by inflammation and degrades type I collagen in thin-cap fibro – atheromata. European Heart Journal 32, 2713–2722.
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10 Xiangdong, L., Yuanwu, L., Hua, Z., Liming, R., Qiuyan, L. & Ning, L. (2011). Animal models for the atherosclerosis research: A review. Protein Cell 2, 189–201.
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20 Wada, Y., Sugiyama, A., Kohro, T., Kobayashi, M., Takeya, M., Naito, M. & Kodama, T. (2000). In vitro model of atherosclerosis using coculture of arterial wall cells and macrophage. Yonsei Medical Journal 41, 740–755.
21 Dorweiler, B., Torzewski, M., Dahm, M., Ochsenhirt, V., Lehr, H.A., Lackner, K.J. & Vahl, C.F.  2006). A novel in vitro model for the study of plaque development in atherosclerosis. Thrombosis & Haemostasis 95, 182–189.
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23 Cummings, I., George, S., Kelm, J., Schmidt, D.,  Emmert, M.Y., Weber, B., Zünd, G., Hoerstrup, S.P. (2012). Tissue-engineered vascular graft remodeling in a growing lamb model: Expression of matrix metalloproteinases. European Journal of Cardiothoracic Surgery 41, 167–172.
24 Hoerstrup, S.P., Cummings, I., Lachat, M., Schoen, F.J., Jenni, R., Leschka, S., Neuenschwander, S., Schmidt, D., Mol, A., Günter, C., Gössi, M., Genoni, M. & Zund, G. (2006). Functional growth in tissueengineered living, vascular grafts: Follow-up at 100 weeks in a large animal model. Circulation 114, Suppl. 1, I159–I166.
25 Kelm, J.M., Emmert, M.Y., Zürcher, A., Schmidt, D., Begus Nahrmann, Y., Rudolph, K.L., Weber, B., Brokopp, C.E., Frauenfelder, T., Leschka, S., Odermatt, B., Jenni, R., Falk, V., Zünd, G. &  Hoerstrup, S.P. (2012). Functionality, growth and accelerated aging of tissue engineered living autologous vascular grafts. Biomaterials 33, 8277–8285.